Compositions and methods related to miRNA modulation of neovascularization or angiogenesis

ABSTRACT

The present invention concerns methods and compositions for diagnosing and/or treating vascular diseases including cancer, cardiac diseases, vascular diseases of the eye, and inflammatory diseases. The methods involve measuring the levels of one or multiple miRNAs in patient samples and using the test results to diagnose and/or predict an optimal treatment regimen for the patient. Compositions described in the invention include nucleic acids that function as miRNAs or miRNA inhibitors that can be introduced to a patient to reduce or increase vascularization as needed.

This application claims priority to U.S. Provisional Application Ser.No. 61/051,519 filed May 8, 2008, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of molecularbiology and medicine. More particularly, it concerns methods andcompositions involving microRNA (miRNAs) molecules and diseasetreatment. Certain aspects of the invention include applications ofmiRNA therapy for diseases or conditions that involve neovascularizationand/or angiogenesis.

II. Background

Pathologic neovascularization refers to the proliferation of bloodvessels in tissue not normally containing them, or proliferation ofblood vessels of a different kind than usual in a tissue. It includesangiogenesis in tumor growth, diabetic retinopathy, haemangiomas,arthritis, and psoriasis to name a few.

Angiogenesis is the process of forming new blood vessels frompre-existing capillaries. Angiogenesis is tightly regulated and normallydoes not occur except during development, wound healing, and theformation of the corpus luteum during the female reproductive cycle.This strict regulation is manifested by a balanced production ofpositive and negative factors, which keep angiogenesis in check.However, this balance becomes abrogated under various pathologicalconditions, such as cancer, diabetes, and age-related maculardegeneration (AMD), resulting in the growth of new blood vessels. It isnow well accepted that the progressive growth and metastasis of manysolid tumors and loss of vision with diabetes are dependent on thegrowth of new blood vessels.

Vascular diseases of the eye and tumors of the central nervous system,such as retinoblastoma and primitive neuroectodermal tumors (PNETs),have significant neovascular components. Some tumors of the centralnervous system and ocular vascular diseases share similar pathogenesishaving a choroidal neovascularization and/or retinal neovascularizationcomponent.

Vascular diseases of the eye comprise a major cause of blindness. Thesediseases include various retinopathies and macular degeneration.Existing treatments include laser ablation of various regions of theretina; vitrectomy or removal of the cloudy vitreous humor and itsreplacement with a saline solution; and administration of antioxidantvitamins E and C, but none of these methods can cure the disease.Further, existing invasive treatment methods often result in significantloss of vision. Non-invasive methods of treatment are experimental andhave not been shown to substantially reduce the risk of blindness orloss of sight.

There is a need for additional compositions and/or methods for treatmentof diseases associated with neovascularization and angiogenesis.

SUMMARY OF THE INVENTION

The present invention provides additional diagnostic, prognostic, and/ortherapeutic methods by identifying miRNAs that are differentiallyexpressed or mis-regulated in various states of diseased, normal,cancerous, and/or abnormal tissues, including but not limited todiseases characterized by or associated with vascularization andangiogenesis that result in a pathological condition or disease.

A physician may choose to treat a condition associated withneovascularization and/or angiogenesis by surgery, chemotherapy,radiation therapy, immunotherapy, monoclonal antibody therapy and/orother methods. The choice of therapy depends upon the location andseverity of the disease, as well as the general state of the patient.Certain aspects of the invention include methods for reducingvascularization in a subject or tissue comprising administering to thesubject or tissue in need of such a reduction, in an amount sufficientto reduce vascularization and/or result in a beneficial response, one ormore nucleic acid molecule comprising (a) a nucleic acid sequence thatis at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toone or more miR-184 (SEQ ID NO:1 and 2), miR-31 (SEQ ID NO:3 and 4),miR-150 (SEQ ID NO:5 and 6), miR-409 (SEQ ID NO:7 and 8), miR-375 (SEQID NO:9 and 10), miR-129-5p (SEQ ID NO:11 and 12), miR-142a (SEQ IDNO:13 and 14), miR-29a (SEQ ID NO:15 and 16), miR-129-3p (SEQ ID NO:17and 18), miR-10b (SEQ ID NO:19 and 20), miR-96 (SEQ ID NO:21 and 22),miR-183 (SEQ ID NO:23 and 24), miR-16 (SEQ ID NO:25 and 26), miR-182(SEQ ID NO:27 and 28), miR-191 (SEQ ID NO:29 and 30), miR-29c (SEQ IDNO:31 and 32), miR-181c (SEQ ID NO:33 and 34), miR-335 (SEQ ID NO:35 and36), miR-7026 (SEQ ID NO:37), miR-210 (SEQ ID NO:38 and 39), miR-512-3p(SEQ ID NO:40 and 41), miR-132 (SEQ ID NO:42 and 43), miR-500 (SEQ IDNO:44 and 45), miR-339 (SEQ ID NO:46 and 47), miR-511 (SEQ ID NO:48 and49), miR-26b (SEQ ID NO:50 and 51), miR-30b (SEQ ID NO:52 and 53), ormiR-15a (SEQ ID NO:54 and 55), or complement thereof, and/or (b) aninhibitor of miR-451 (SEQ ID NO:56 and 57), miR-424 (SEQ ID NO:58 and59), miR-146 (SEQ ID NO:60 and 61), miR-214 (SEQ ID NO:62 and 63),miR-199a (SEQ ID NO:64 and 65), miR-181 (SEQ ID NO:66 and 67), miR-350(SEQ ID NO:68 and 69), miR-21 (SEQ ID NO:70 and 71), miR-218 (SEQ IDNO:72 and 73), miR-148b (SEQ ID NO:74 and 75), miR-106a (SEQ ID NO:76and 77), miR-205 (SEQ ID NO:78 and 79), miR-365 (SEQ ID NO:80 and 81),miR-299-5p (SEQ ID NO:82 and 83), ambi-miR-7079 (SEQ ID NO:84), miR-200a(SEQ ID NO:85 and 86), miR-351 (SEQ ID NO:87 and 88), miR-329 (SEQ IDNO:89 and 90), miR-122a (SEQ ID NO:91 and 92), miR-20a (SEQ ID NO:93 and94), miR-520h (SEQ ID NO:95 and 96), miR-142-5p (SEQ ID NO:97 and 98),miR-203 (SEQ ID NO:99 and 100), miR-211 (SEQ ID NO:101 and 102), miR-145(SEQ ID NO:103 and 104), let-7b (SEQ ID NO:105 and 106), miR-93 (SEQ IDNO:107 and 108), miR-192 (SEQ ID NO:109 and 110), miR-201 (SEQ ID NO:111and 112), miR-18a (SEQ ID NO:113 and 114), miR-17-5p (SEQ ID NO:115 and116), miR-7085 (SEQ ID NO:117), miR-106b (SEQ ID NO:118 and 119), ormiR-223 (SEQ ID NO:120 and 121). In certain aspects, a nucleic acid isadministered topically, enterally, parenterally or intravitreally. In afurther aspect, the nucleic acid molecule is an RNA, DNA, or comprisesall or portions of nucleotide analogs or mimetics. An RNA can comprise acomplementary RNA region, such as, but not limited to a hairpinstructure or multiple (e.g., two) RNA strands. The RNA or DNA moleculecan include a nucleotide analog or a modified nucleotide. In still afurther aspect, the nucleic acid molecule or miRNA inhibitor is a DNAmolecule or is produced from a DNA molecule. An miRNA inhibitor can bean antisense oligonucleotide. The oligonucleotide can comprise all or aportion of nucleotide analogs. In other aspects the DNA is comprised inan expression cassette, such as a plasmid expression vector or a viralexpression vector. The nucleic acid molecule can also be comprised in alipid or viral delivery vehicle.

In certain aspects the subject has, is at risk of developing, or issuspected of having ocular or retinal/choroidal neovascular diseases,cancer, diabetic nephropathy, rheumatoid arthritis, atheroscleroticplaques, endometriosis, Crohn's disease, uterine fibroids, benignprostatic hyperplasia, or psoriasis. The nucleic acids or nucleic acidanalogs disclosed herein are useful for treating and/or preventing avariety of angiogenic, microvascular, and macular disorders includingmacular degeneration (such as wet or neovascular age-related maculardegeneration (AMD) and dry or atrophic AMD), macular edema, andsecondary indications for inhibiting tumor vascularization, and cornealand iris neovascularization, for example.

A further aspect of the invention includes methods of stimulatingvascularization in a subject or tissue comprising administering to asubject in need of such stimulation, in an amount sufficient tostimulate vascularization, one or more nucleic acid molecule comprising(a) a miRNA sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% identical to one or more of miR-451, miR-424, miR-146,miR-214, miR-199a, miR-181, miR-350, miR-21, miR-218, miR-148b,miR-106a, miR-205, miR-365, miR-299-5p, ambi-miR-7079, miR-200a,miR-351, miR-329, miR-122a, miR-20a, miR-520h, miR-142-5p, miR-203,miR-211, miR-145, let-7b, miR-93, miR-192, miR-201, miR-18a, miR-17-5p,miR-7085, miR-106b, and/or miR-223; and/or (b) an inhibitor of miR-184,miR-31, miR-150, miR-409, miR-375, miR-129-5p, miR-142a, miR-29a,miR-129-3p, miR-10b, miR-96, miR-183, miR-16, miR-182, miR-191, miR-29c,miR-181c, miR-335, miR-7026, miR-210, miR-512-3p, miR-132, miR-500,miR-339, miR-511, miR-26b, miR-30b, and/or miR-15a. In further aspectsof the invention the subject has, is at risk of developing, or issuspected of having coronary artery disease (CAD), cardiac failure,tissue injury, or ischemia.

In yet another aspect the invention includes methods for selecting avascular therapy for a patient comprising (a) measuring an expressionprofile of one or more of miR-184, miR-31, miR-150, miR-409, miR-375,miR-129-5p, miR-142a, miR-29a, miR-129-3p, miR-10b, miR-96, miR-183,miR-16, miR-182, miR-191, miR-29c, miR-181c, miR-335, miR-7026, miR-210,miR-512-3p, miR-132, miR-500, miR-339, miR-511, miR-26b, miR-30b,miR-15a, miR-451, miR-424, miR-146, miR-214, miR-199a, miR-181, miR-350,miR-21, miR-218, miR-148b, miR-106a, miR-205, miR-365, miR-299-5p,ambi-miR-7079, miR-200a, miR-351, miR-329, miR-122a, miR-20a, miR-520h,miR-142-5p, miR-203, miR-211, miR-145, let-7b, miR-93, miR-192, miR-201,miR-18a, miR-17-5p, miR-7085, miR-106b, and/or miR-223 in a sample; and(b) selecting a therapy based on a comparison of the miRNA expressionprofile in the patient sample to an expression profile of a normal ornon-pathogenic sample, wherein a difference between the expressionprofiles is indicative of a pathological condition. The alteredexpression for any of the miRNAs indicates that the patient should betreated with a corresponding therapeutic directed toward the alteredmiRNA or condition indicated by such altered miRNA.

In some embodiments, it may be useful to know whether a cell expresses aparticular miRNA endogenously or whether such expression is affectedunder particular conditions or when it is in a particular disease state.Thus, in some embodiments of the invention, methods include assaying acell or a sample containing a cell for the presence of one or moremiRNA. Consequently, in some embodiments, methods include a step ofgenerating a miRNA profile for a sample. The term “miRNA profile” refersto data regarding the expression pattern of miRNAs in the sample (e.g.,one or more miRNA described herein). It is contemplated that the miRNAprofile can be obtained using a set of miRNAs, using for example nucleicacid amplification or hybridization techniques well known to one ofordinary skill in the art. In certain embodiments, expression of one ormore miRNA described herein is evaluated.

In some embodiments of the invention, an miRNA profile is generated bysteps that include one or more of: (a) labeling miRNA in the sample; (b)hybridizing miRNA to a number of probes, or amplifying a number ofmiRNAs, and/or (c) determining miRNA hybridization to the probes ordetecting miRNA amplification products, wherein miRNA expression levelsare determined or evaluated. See U.S. Provisional Patent Applications60/575,743 and 60/649,584, and U.S. patent application Ser. Nos.11/141,707 and 11/855,792, all of which are hereby incorporated byreference.

Methods of the invention include determining a diagnosis or prognosisfor a patient based on miRNA expression or expression levels. In certainembodiments, the elevation or reduction in the level of expression of aparticular miRNA or set of miRNAs in a cell is correlated with a diseasestate as compared to the expression level of that miRNA or set of miRNAsin a normal cell or a reference sample or digital reference. Thiscorrelation allows for diagnostic methods to be carried out when theexpression level of a miRNA is measured in a biological sample beingassessed.

In certain embodiments a method for evaluating a patient includes thesteps of determining expression levels of one or more of miR-451,miR-424, miR-146, miR-214, miR-199a, miR-181, miR-350, miR-21, miR-218,miR-148b, miR-106a, miR-205, miR-365, miR-299-5p, ambi-miR-7079,miR-200a, miR-351, miR-329, miR-122a, miR-20a, miR-520h, miR-142-5p,miR-203, miR-211, miR-145, let-7b, miR-93, miR-192, miR-201, miR-18a,miR-17-5p, miR-7085, miR-106b, miR-223, miR-184, miR-31, miR-150,miR-409, miR-375, miR-129-5p, miR-142a, miR-29a, miR-129-3p, miR-10b,miR-96, miR-183, miR-16, miR-182, miR-191, miR-29c, miR-181c, miR-335,miR-7026, miR-210, miR-512-3p, miR-132, miR-500, miR-339, miR-511,miR-26b, miR-30b, and/or miR-15a in a biological sample comprising aportion of a tissue or fluid associated with a condition associated withaberrant vascularization, and/or determining a diagnosis or prognosisfor the aberrant vascularization condition based on the miRNA expressionlevels. In a further aspect of the invention, a patient is suspected ofhaving a condition associated with aberrant vascularization. Determininga diagnosis includes, but is not limited to screening for a pathologicalcondition, staging a pathological condition, or assessing response of apathological condition to therapy. In certain aspects, determining adiagnosis is determining if the patient has a condition associated withaberrant neovascularization or aberrant angiogenesis.

Methods can further comprise normalizing the expression levels of miRNA.Normalizing includes, but is not limited to adjusting expression levelsof miRNA relative to expression levels of one or more nucleic acid inthe sample.

It is specifically contemplated that miRNA profiles for patients,particularly those suspected of having or at risk of developing aparticular disease or condition associated or related to vascularizationand/or angiogenesis, can be generated by evaluating any miR or set ofmiRs discussed in this application. The miRNA profile that is generatedfrom the patient will be one that provides information regarding theparticular disease or condition. In certain aspects, a party evaluatingmiR expression may prepare a recommendation, report and/or summaryconveying processed or raw data to a diagnosing physician. In certainaspects, a miRNA profile can be used in conjunction with otherdiagnostic tests.

Embodiments of the invention include methods for diagnosing, assessing acondition, and/or prognosing a disease or condition associated with orhaving an accompanying aberrant vascularization in a patient comprisingevaluating or determining the expression or expression levels of one ormore miRNAs in a sample from the patient. The difference in theexpression in the sample from the patient and a reference, such asexpression in a normal or non-pathologic sample, is indicative of apathologic or diseased condition associated with neovascularizationand/or angiogenesis. In certain aspects the miRNA expression level iscompared to the expression level of a normal cell or a reference sampleor a digital reference. Comparing miRNA expression levels includescomparing miRNA expression levels in a sample to miRNA expression levelsin a normal tissue sample or reference tissue sample. A normal tissuesample can be taken from the patient being evaluated and can be a normaladjacent tissue to the area being assessed or evaluated.

In certain embodiments the expression of the miRNA is determined by anamplification assay or a hybridization assay. An amplification assay isa quantitative amplification assay, such as, but not limited toquantitative RT-PCR. Hybridization assays include, but are not limitedto an array hybridization assay or a solution hybridization assay. AnmiRNA, amplification product, or probe set can comprise a segment of orbe complementary to a corresponding miRNA including all or part of anmiRNA sequence described herein. In certain aspects of the invention, asegment can comprise at least or about 5, 6, 7, 8, 9, 10, 11, 12 or morenucleic acid sequences of a miRNA. Other amplification or hybridizationsequences may also be included for normalization purposes. The use of anmiRNA quantification assay as a clinically relevant diagnostic tool canbe enhanced by using an appropriate normalization control. The methodsof normalization correct for sample-to-sample variability by comparing atarget measurement in a sample to one or more internal controls.Normalization of miRNA quantification assays reduces systematic(non-biological) and non-systematic differences between samples, and canenhance the accurate measurement of differential miRNA expression, forexample. The accurate measurement of biologically hardwired differentialexpression between two groups of samples is the goal of many miRNAqRT-PCR assays. Yet, miRNA levels in qRT-PCR reactions can vary from onesample to the next for reasons that may be technical or biological.Technical reasons may include variability in tissue procurement orstorage, inconsistencies in RNA extraction or quantification, ordifferences in the efficiency of the reverse transcription and/or PCRsteps. Biological reasons may include sample-to-sample heterogeneity incellular populations, differences in bulk transcriptional activity, oralterations in specific miRNA expression that is linked to an aberrantbiological program (e.g., a disease state). Given the multiplicity ofsources that can contribute to differences in miRNA quantification,results from qRT-PCR assays can be normalized against a relevantendogenous target or targets to minimize controllable variation, andpermit definitive interpretations of nominal differences in miRNAexpression.

Because conditions related to neovascularization and/or angiogenesis,such as cancer, refer to a class of diseases, it is unlikely that therewill be a single treatment and aspects of the invention can be used todetermine which treatment will be most effective or most harmful andprovide a guide for the physician in evaluating, assessing, andformulating a treatment strategy for a patient. A sample may be takenfrom a patient having or suspected of having a disease or pathologicalcondition. In certain aspects, the sample can be, but is not limited totissue (e.g., biopsy, particularly fine needle biopsy), sputum, lavagefluid, blood, serum, plasma, lymph node or other tissue or fluid thatmay contain cells associated with a neovascular or angiogenic condition.The sample can be fresh, frozen, fixed (e.g., formalin fixed), orembedded (e.g., paraffin embedded).

The methods can further comprise or exclude one or more steps including:(a) obtaining a sample from the patient, (b) isolating or obtainingnucleic acids from the sample, (c) reverse transcribing nucleic acidsfrom the sample, (d) labeling the nucleic acids isolated from the sampleor an amplification product thereof, (e) hybridizing the labeled nucleicacids to one or more probes or detecting the amplified nucleic acids,(f) analyzing and normalizing data by statistical methods, and/or (g)creating and/or supplying a report of the analysis. Nucleic acids of theinvention may include one or more nucleic acids comprising at least onesegment having a sequence or complementary sequence of one or more miRNAdescribed herein. In certain aspects, the nucleic acids identify one ormore miRNA described herein. Nucleic acids of the invention may becoupled to a support. Such supports are well known to those of ordinaryskill in the art and include, but are not limited to glass, plastic,metal, or latex. In particular aspects of the invention, the support canbe planar or in the form of a bead or other geometric shapes orconfigurations.

Embodiments of the invention include kits for analysis of a pathologicalsample by assessing a miRNA profile for a sample comprising, in suitablecontainer means, one or more miRNA probes and/or amplification primers,wherein the miRNA probes detect or primer amplify one or more miRNAdescribed herein. In certain embodiments the kit contains two or moremiRNA hybridization or amplification reagents comprising one or moreprobe or amplification primer for one or more miRNA selected frommiR-451, miR-424, miR-146, miR-214, miR-199a, miR-181, miR-350, miR-21,miR-218, miR-148b, miR-106a, miR-205, miR-365, miR-299-5p,ambi-miR-7079, miR-200a, miR-351, miR-329, miR-122a, miR-20a, miR-520h,miR-142-5p, miR-203, miR-211, miR-145, let-7b, miR-93, miR-192, miR-201,miR-18a, miR-17-5p, miR-7085, miR-106b, miR-223, miR-184, miR-31,miR-150, miR-409, miR-375, miR-129-5p, miR-142a, miR-29a, miR-129-3p,miR-10b, miR-96, miR-183, miR-16, miR-182, miR-191, miR-29c, miR-181c,miR-335, miR-7026, miR-210, miR-512-3p, miR-132, miR-500, miR-339,miR-511, miR-26b, miR-30b, and/or miR-15a. The kit can further comprisereagents for labeling miRNA in the sample. The kit may also includelabeling reagents comprising at least one amine-modified nucleotide,poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents caninclude an amine-reactive dye.

The present invention also concerns kits containing compositions of theinvention or compositions to implement methods of the invention. In someembodiments, kits can be used to evaluate one or more miRNA molecules.In certain embodiments, a kit contains, contains at least or contains atmost 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more miRNA probes,synthetic miRNA molecules or miRNA inhibitors, or any range andcombination derivable therein. In some embodiments, there are kits forevaluating miRNA activity in a cell.

Kits may comprise components, which may be individually packaged orplaced in a container, such as a tube, bottle, vial, syringe, or othersuitable container means.

Individual components may also be provided in a kit in concentratedamounts; in some embodiments, a component is provided individually inthe same concentration as it would be in a solution with othercomponents. Concentrations of components may be provided as 1×, 2×, 5×,10×, or 20× or more, including all values and ranges there between.

Kits for using miRNA probes, synthetic miRNAs, nonsynthetic, and/ormiRNA inhibitors of the invention for therapeutic, prognostic, ordiagnostic applications are included as part of the invention.

The term “miRNA” or “miR” is used according to its ordinary and plainmeaning and refers to a microRNA molecule found in eukaryotes that isinvolved in RNA-based gene regulation. See, e.g., Carrington et al.,2003, which is hereby incorporated by reference. The term will be usedto refer to the single-stranded RNA molecule processed from a precursor.Names of miRNAs and their sequences related to the present invention areprovided herein.

Corresponding miRNA sequences that can be used in the context of theinvention include, but are not limited to, all or a portion of thosesequences in the sequence listing provided herein, as well as the miRNAprecursor sequence, or complement of one or more of these miRNAs.

Any embodiment of the invention involving specific miRNAs by name iscontemplated also to cover embodiments involving miRNAs whose sequencesare at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99% identical to the mature sequence of the specifiedmiRNA. In other aspects, miRNA of the invention may include additionalnucleotides at the 5′, 3′, or both 5′ and 3′ ends of at least, at mostor about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein and that different embodiments may be combined. It isspecifically contemplated that any methods and compositions discussedherein with respect to miRNA molecules or miRNA may be implemented withrespect to synthetic miRNAs. Typically, the synthetic miRNA is exposedto the proper conditions to allow it to become or function, at least inpart, as a mature miRNA under physiological circumstances.

The claims originally filed are contemplated to cover claims that aremultiply dependent on any filed claim or combination of filed claims.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Any embodiment discussed with respect to a particularcondition can be applied or implemented with respect to a differentcondition. Furthermore, compositions and kits of the invention can beused to achieve methods of the invention.

Throughout this application, the term “about” may be used to indicatethat a value includes the standard deviation of error for the device ormethod being employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1B. QRT-PCR quantification of mmu-miR-31, mmu-miR-150, andmmu-miR-184 in mouse retinas undergoing neovascularization. Meanthreshold cycle numbers (ΔCT) were determined for RNA from ischemic andcontrol retinas (n=5 each) and the mean difference (ΔΔCT) ranged fromabout −1.0 (miR-31) to −2.5 (miR-150) (FIG. 1A). Statistical comparisonsconfirmed that the quantity of each miRNA was significantly reduced inischemic compared to control retinas (FIG. 1B).

FIG. 2. Putative binding regions for mmu-miR-31, -150, and -184 in3′-untranslated regions (UTRs) of predicted target mRNAs (SEQ IDNOS:122-134).

FIGS. 3A-3F. Effect of different microRNAs on luciferase activity fromreporter genes harboring putative miRNA binding sites. Luciferasereporter assays were performed as described in Example 4. Each barrepresents the mean luciferase activity (±standard error of mean)calculated from three experiments. pMIR-Luc-Pdgfb-3′UTR (FIGS. 3A and3D); pMIR luciferase reporter vector containing 3′-UTR from Pdgfb.pMIR-Luc-HIF1α-3′UTR (FIG. 3C); pMIR luciferase reporter vectorcontaining 3′-UTR from Hif1α. pMIR-Luc-Fz4-3′UTR (FIG. 3B); pMIRluciferase reporter vector containing 3′-UTR from Frizzled4.pGL2-Luc-Vegf-3′UTR (FIG. 3E); pGL2-CMV luciferase reporter vectorcontaining 3′-UTR from Vegf. pMIR-Luc-Notch4-3′UTR (FIG. 3F); pMIRluciferase reporter vector containing 3′-UTR from Notch4.

FIGS. 4A-4B. Effect of microRNAs on target gene product levels inneovascularizing retinas in vivo. (FIG. 4A) VEGF levels in homogenatesof experimental miRNA-treated and control miRNA-treated retinas,measured by ELISA. Each bar represents the mean VEGF level (±standarderror of mean) calculated from three separate experiments. Asterisksindicate levels of VEGF significantly different from control-treatedretinas. (FIG. 4B) HIF-1α, PDGFB, VEGF, and Frizzled4 levels inhomogenates of experimental miRNA-treated control miRNA-treated retinas,measured by immunoblot analysis. Each immunoblot was repeated at leastonce with similar results.

FIGS. 5A-5B. Neovascularization in retinas from mouse eyes followingischemia-induced retinopathy. FIG. 5A. Mouse eyes were injected withnegative control miRNA. FIG. 5B. Mouse eyes were injected with a mixtureof synthetic mmu-miR-31, -150, and -184. Brightly fluorescent areas areareas of neovascularization.

FIGS. 6A-6D. Effect of microRNAs on ischemia-induced retinalneovascularization. Each bar represents the mean area ofneovascularization (±standard deviation) calculated from eight mice.Statistical comparisons were made by paired t-test.

FIGS. 7A-7B. Choroidal neovascularization at Bruch's membrane rupturesites following laser photocoagulation. (FIG. 7A) Mouse eyes wereinjected with negative control miRNA. (FIG. 7B) Mouse eyes were injectedwith a mixture of synthetic mmu-miR-31, -150, and -184. Brightlyfluorescent areas are areas of neovascularization.

FIGS. 8A-8D. Effect of microRNAs on choroidal neovascularization atBruch's membrane rupture sites in a mouse model. Each bar represents themean area of choroidal neovascularization (mm³±standard deviation)calculated from at least twelve experimental values. Statisticalcomparisons were made by paired t-test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods relatingto preparation and characterization of miRNAs, as well as use of miRNAsfor therapeutic, prognostic, and/or diagnostic applications,particularly those methods and compositions related to assessing and/oridentifying conditions associated with aberrant or abnormalvascularization and/or angiogenesis.

I. NEOVASCULARIZATION AND ANGIOGENESIS

Neovascularization refers to the proliferation of blood vessels that areatypical for tissues containing them or the proliferation of bloodvessels distinct from those that are normally found in the tissue.Neovascularization occurs through a complex process called angiogenesis.Angiogenesis is regulated by a mixture of stimulators and inhibitorsthat are balanced to ensure the proper development of blood vessels inthe body. Mis-regulated angiogenesis is the underlying cause of numerousdiseases and a contributor to many more.

Inhibition of angiogenesis can be a useful therapy for diseases such asdiabetic nephropathy, rheumatoid arthritis, atherosclerotic plaques,endometriosis, Crohn's disease, uterine fibroids, benign prostatichyperplasia, psoriasis, and cancer. In 1988, interferon α2a was firstused as an antiangiogenic drug to treat children with life-threateninghemangiomas, a nonmalignant vascular tumor (White et al., 1989; Folkman,1989). Several hundred angiogenesis inhibitors have since beendiscovered and many of these have been evaluated for therapeutic utilityin clinical trials. Many of these compounds have not proved useful fordisease therapy. However, some cancer patients have experienced dramaticregression of their tumors from antiangiogenic therapy, and others haveexperienced stabilization of their disease. The first FDA-approved bloodvessel therapy for eye disease was Visudyne (QLTTherapeutics/CibaVision), which has shown effectiveness for treatingmacular degeneration. Additional angiogenesis inhibitors have since beenapproved for the treatment of macular degeneration.

In contrast, stimulation of angiogenesis can be a useful therapy for thetreatment of coronary artery disease (CAD), cardiac failure, tissueinjury, and ischemic diseases such as ischemic CAD, critical limbischemia with various etiologies, and decubitus. The first FDA-approveddevice to stimulate growth of new blood vessels in diseased hearts was alaser used in a technique referred to as direct myocardialrevascularization (DMR) or transmyocardial revascularization (TMR). Thefirst angiogenesis-stimulating medicine was a prescription gel calledRegranex (recombinant human platelet-derived growth factor-BB,Ortho-McNeil Pharmaceuticals) that is approved for treatment of diabeticfoot ulcers. Numerous angiogenic growth factors and angiogenic genetherapies are now being developed or tested in humans for growing newblood vessels to heal wounds and for restoring blood flow to the heart,limbs, and brain.

Although differences exist among vascular beds throughout the body, thegenes and molecules involved in stimulating or inhibitingneovascularization tend to be common. Animal models for retinal andchoroidal neovascularization (CNV) have proved useful in understandingthe molecular events leading to blood vessel formation (Campochiaro andHackett, 2003). Consistent with neovascularization in other tissues,retinal and choroidal neovascularization is stimulated by vascularendothelial growth factor (VEGF) (Aiello et al., 1995; Miller et al.,1994; Ozaki et al., 2000; Kwak et al., 2000). Intraocular injections ofranibizumab, an Fab that binds all isoforms of VEGF-A, resulted insignificant visual improvement in 35-40% of patients with CNV caused byage-related macular degeneration (AMD) (Rosenfeld et al., 2006; Brownand Regillo, 2007). While this has benefited many patients with CNV, theremaining 60-65% of CNV patients will need another type of treatment torealize vision improvement.

Transcriptional regulation of many genes is clearly important fordetermining the balance of stimulators and inhibitors ofneovascularization. In addition, mounting evidence now suggests thatmicroRNAs may play a complementary role in regulation ofneovascularization. MicroRNAs (miRNAs) are small non-coding RNAs,comprising an evolutionarily conserved class of regulatory nucleic acidsranging in size from 14-35 nucleotides in their mature form. miRNAprecursors are processed by cellular proteins, including Drosha andDicer, to generate a short double-stranded miRNA. One of the miRNAstrands is incorporated into a complex of proteins and miRNA called theRNA-induced silencing complex (RISC). The miRNA guides the RISC complexto a target mRNA, which is then cleaved or translationally silenced,depending on the degree of sequence complementarity of the miRNA to itstarget mRNA (Bagga et al, 2005; Lim et al., 2005). In mice, targeteddisruption of Dicer, results in death during the embryonic growth phase,due to disruption of vascular development (Yang et al., 2005). Morerecently, two miRNAs (miR-221 and miR-222) have been shown to modulatethe angiogenic properties of human umbilical vein endothelial cells(Poliseno et al., 2006).

Compositions and methods of the present invention can be used to inhibitneovascularization. Accordingly, the present invention discloses methodsof using miRNA or miRNA inhibitor for treating a variety of diseaseswith a neovascularization component (hereinafter collectively referredto as “neovascularization diseases” or “conditions associated withaberrant vascularization or angiogenesis”) or other similar phrases. Inone embodiment, the present invention provides a method of treatingpathological conditions resulting from angiogenesis orneovascularization comprising administration of an effective amount ofan miRNA or an miRNA inhibitor.

Diseases of the eye treatable using the present invention include, butare not limited to retinopathy of prematurity (ROP), age-related maculardegeneration (AMD), diabetic retinopathy, hypertensive retinopathy,central retinal vein occlusion (CRVO), branch vein occlusion (BRVO),neovascular glaucoma, ocular ischemic syndrome, occlusive vasculitis,polypoidal choroidal vasculopathy, myopic choroidal neovascularization,radiation retinopathy, chorioretinitis, central serous choroidopathy,central retinal artery occlusion, uveitic macular edema, idiopathicjuxtafoveal telangiectasia, angioid streaks, sickle cell retinopathy,and pseudophakic cystoid macular edema. All of these diseases share thesame mechanism of choroidal neovascularization and/or retinalneovascularization, the inhibition of which will result in treatment ofthe diseases.

Other diseases of the eye treatable by the present invention include,but are not limited to, primary ocular tumors, such as uveal melanomas,melanocytomas, retinocytomas, retinal hamartomas and choristomas,retinal angiomas, retinal gliomas and astrocytomas, choroidalhemangiomas, choroidal neurofibromas, choroidal hamartomas andchoristomas, ocular lymphomas and ocular phakomatoses; and metastaticocular tumors related to choroidal and retinal neovascularization.

In addition, the present invention is suitable for the treatment ofcancers such as, but not limited to medulloblastoma, pineoblastoma,non-pineal supratententorial, and Ewings sarcoma.

II. miRNA BASED THERAPY

Embodiments of the invention concern nucleic acids that perform theactivities of or inhibit endogenous miRNAs when introduced into cells.In certain aspects, nucleic acids are synthetic or non-synthetic miRNA.Sequence-specific miRNA or miRNA inhibitors can be used to inhibitsequentially or in combination the activities of one or more endogenousmiRNAs in cells, as well those genes and associated pathways modulatedby the endogenous miRNA.

Methods of the invention include supplying or enhancing the activity ofone or more miRNAs in a cell. The present invention also concernsinducing certain cellular characteristics by providing to a cell aparticular nucleic acid, such as a specific synthetic miRNA molecule.However, in methods of the invention, the miRNA molecule or miRNAinhibitor need not be synthetic. They may have a sequence that isidentical to a naturally occurring miRNA or they may not have any designmodifications. In certain embodiments, the miRNA molecule is synthetic,as discussed herein.

The particular nucleic acid molecule provided to the cell is understoodto correspond to a particular miRNA in the cell, and thus, the miRNA inthe cell is referred to as the “corresponding miRNA.” In situations inwhich a named miRNA molecule is introduced into a cell, thecorresponding miRNA will be understood to be the induced or inhibitedmiRNA or induced or inhibited miRNA function. It is contemplated,however, that the miRNA molecule introduced into a cell is not a maturemiRNA but is capable of becoming or functioning as a mature miRNA underthe appropriate physiological conditions. It is contemplated thatmultiple corresponding miRNAs may be involved. A miRNA may have aminimal adverse effect on a subject or patient while supplying asufficient therapeutic effect, such as amelioration of a condition,growth inhibition of a cell, death of a targeted cell, alteration ofcell phenotype or physiology, slowing of cellular growth, sensitizationto a second therapy, sensitization to a particular therapy, and thelike. Methods include identifying a cell or patient in need of inducingthose cellular characteristics. Also, it will be understood that anamount of a synthetic nucleic acid that is provided to a cell ororganism is an “effective amount” or “amount sufficient” for aparticular result, which refers to an amount needed (or a sufficientamount) to achieve a desired goal, such as inducing a particularcellular characteristic(s), inhibiting vascularization or stimulatingvascularization. In certain embodiments of the methods include providingor introducing to a cell a nucleic acid molecule corresponding to amature miRNA in the cell in an amount effective to achieve a desiredphysiological result. Moreover, methods can involve providing syntheticor nonsynthetic miRNA molecules. Furthermore, any method articulatedusing a list of miRNAs using Markush group language may be articulatedwithout the Markush group language and a disjunctive article (i.e., or)instead, and vice versa.

In some embodiments, there is a method for reducing or inhibitingvascularization comprises introducing into or providing a subject,tissue, or cell an effective amount of a synthetic or nonsynthetic miRNAmolecule that corresponds to a miRNA sequence disclosed herein, or acomplement or inhibitor thereof.

Certain embodiments of the invention include methods of treating apathologic condition. In one aspect, the method comprises contacting atarget cell with one or more nucleic acid, synthetic miRNA, or miRNAcomprising at least one nucleic acid segment having all or a portion ofa miRNA sequence. The segment may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides ornucleotide analog including all integers there between. In certainaspects, one or more nucleotide of a nucleic acid can be modified. Anaspect of the invention includes the modulation of gene expression,miRNA expression or function, or mRNA expression or function within atarget subject, tissue, or cell.

Typically, an endogenous gene, miRNA or mRNA is modulated in the cell.In particular embodiments, the nucleic acid sequence comprises at leastone segment that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or100% identical in nucleic acid sequence to one or more miRNA or genesequence. Modulation of the expression or processing of an endogenousgene, miRNA, or mRNA can be through modulation of the processing of amRNA, such processing including transcription, transportation and/ortranslation in a cell. Modulation may also be effected by the inhibitionor enhancement of miRNA activity in a cell, tissue, or organ. Suchprocessing may affect the expression of an encoded product or thestability of the mRNA. In still other embodiments, a nucleic acidsequence can comprise a modified nucleic acid sequence. In certainaspects, one or more miRNA sequence may include or comprise a modifiednucleobase or nucleic acid sequence.

It will be understood in methods of the invention that a cell or otherbiological matter such as an organism (including patients or subjects)can be provided a miRNA or miRNA molecule corresponding to a particularmiRNA by administering to the cell or organism a nucleic acid moleculethat functions as the corresponding miRNA once inside the cell. Incertain embodiments, it is specifically contemplated that the miRNAmolecule provided to the biological matter is not a mature miRNAmolecule but a nucleic acid molecule that can be processed into themature miRNA once it is accessible to miRNA processing machinery. Theterm “nonsynthetic” in the context of miRNA means that the miRNA is not“synthetic,” as defined herein. Furthermore, it is contemplated that inembodiments of the invention that concern the use of synthetic miRNAs,the use of corresponding nonsynthetic miRNAs is also considered anaspect of the invention, and vice versa. It will be understand that theterm “providing” an agent is used to include “administering” the agentto a patient.

In certain methods of the invention, there is a further step ofadministering the selected miRNA modulator to a cell, tissue, organ, ororganism (collectively “biological matter”) in need of treatment relatedto modulation of the targeted miRNA or in need of the physiological orbiological results discussed herein (such as with respect to aparticular cellular pathway or result like inhibition of vascularizationor angiogenesis, or stimulation or promotion of vascularization orangiogenesis). Consequently, in some methods of the invention there is astep of identifying a patient in need of treatment that can be providedby the miRNA modulator(s). It is contemplated that an effective amountof a miRNA modulator can be administered in some embodiments. Inparticular embodiments, there is a therapeutic benefit conferred on thebiological matter, where a “therapeutic benefit” refers to animprovement in the one or more conditions or symptoms associated with adisease or condition or an improvement in the prognosis, duration, orstatus with respect to the disease. It is contemplated that atherapeutic benefit includes, but is not limited to, a decrease in pain,a decrease in morbidity, a decrease in a symptom. For example, withrespect to cancer, it is contemplated that a therapeutic benefit can beinhibition of tumor growth, prevention of metastasis, reduction innumber of metastases, inhibition of cancer cell proliferation, inductionof cell death in cancer cells, inhibition of angiogenesis near cancercells, induction of apoptosis of cancer cells, reduction in pain,reduction in risk of recurrence, induction of chemo- or radiosensitivityin cancer cells, prolongation of life, and/or delay of death directly orindirectly related to cancer.

Furthermore, it is contemplated that the miRNA compositions may beprovided as part of a therapy to a patient, in conjunction withtraditional therapies or preventative agents. Moreover, it iscontemplated that any method discussed in the context of therapy may beapplied as a preventative measure, particularly in a patient identifiedto be potentially in need of the therapy or at risk of the condition ordisease for which a therapy is needed.

A nucleic acid of the invention can enhance the effect or efficacy of adrug, reduce any side effects or toxicity, modify its bioavailability,and/or decrease the dosage or frequency needed. In certain embodiments,the therapeutic drug is a anti-vascularization, pro-vascularization,and/or a cancer therapeutic. Consequently, in some embodiments, there isa method of treating a patient comprising administering to the patientthe therapeutic and an effective amount of a miRNA molecule thatimproves the efficacy of a second therapeutic or protects non-targetedcells from a detrimental affect of a drug. Therapies also include avariety of combination therapies with both chemical and radiation basedtreatments. Combination chemotherapies include but are not limited to,for example, 5-fluorouracil, alemtuzumab, amrubicin, bevacizumab,bleomycin, bortezomib, busulfan, camptothecin, capecitabine, cisplatin(CDDP), carboplatin, cetuximab, chlorambucil, cisplatin (CDDP), EGFRinhibitors (gefitinib and cetuximab), procarbazine, mechlorethamine,cyclophosphamide, camptothecin, COX-2 inhibitors (e.g., celecoxib),cyclophosphamide, cytarabine) ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, dasatinib, daunorubicin,dexamethasone, docetaxel, doxorubicin (adriamycin), EGFR inhibitors(gefitinib and cetuximab), erlotinib, estrogen receptor binding agents,bleomycin, plicomycin, mitomycin, etoposide (VP16), everolimus,tamoxifen, raloxifene, estrogen receptor binding agents, taxol,taxotere, gemcitabien, navelbine, farnesyl-protein transferaseinhibitors, gefitinib, gemcitabine, gemtuzumab, ibritumomab, ifosfamide,imatinib mesylate, larotaxel, lapatinib, lonafarnib, mechlorethamine,melphalan, transplatinum, 5-fluorouracil, vincristin, vinblastin andmethotrexate, mitomycin, navelbine, nitrosurea, nocodazole, oxaliplatin,paclitaxel, plicomycin, procarbazine, raloxifene, rituximab, sirolimus,sorafenib, sunitinib, tamoxifen, taxol, taxotere, temsirolimus,tipifarnib, tositumomab, transplatinum, trastuzumab, vinblastin,vincristin, or vinorelbine or any analog or derivative variant of theforegoing.

III. NUCLEIC ACIDS

The present invention concerns nucleic acids, modified nucleic acids,nucleic acid mimetics, miRNAs, and segments thereof that can be employedin therapeutic applications, particularly those applications related topathological conditions. The molecules may have been endogenouslyproduced by a cell and isolated, or synthesized or produced chemicallyor recombinantly. They may be isolated and/or purified. Each of themiRNAs described herein includes the corresponding SEQ ID NO andaccession numbers for these miRNA sequences. The name of a miRNA isoften abbreviated and referred to without a “hsa-” prefix and will beunderstood as such, depending on the context. Unless otherwiseindicated, miRNAs referred to in the application are human sequencesidentified as miR-X or let-X, where X is a number and/or letter.

In certain aspects, a miRNA designated by a suffix “5P” or “3P” can beused. “5P” indicates that the mature miRNA derives from the 5′ end ofthe precursor and a corresponding “3P” indicates that it derives fromthe 3′ end of the precursor, as described on the world wide web atsanger.ac.uk. Moreover, in some embodiments, a miRNA probe is used thatdoes not correspond to a known human miRNA. It is contemplated thatthese non-human miRNA probes may be used in embodiments of the inventionor that there may exist a human miRNA that is homologous to thenon-human miRNA. In other embodiments, any mammalian cell, biologicalsample, or preparation thereof may be employed.

The present invention concerns, in some embodiments, short nucleic acidmolecules that function as miRNAs in a cell. The term “short” refers toa length of a single polynucleotide that is at least, at most, or about6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 50, 100, or 150 nucleotides, including all integers or rangesderivable there between. The nucleic acid molecules are typicallysynthetic. The term “synthetic” refers to a nucleic acid molecule thatis isolated and not produced naturally in a cell. In certain aspects thesequence (the entire sequence) and/or chemical structure deviates from anaturally-occurring nucleic acid molecule, such as an endogenousprecursor miRNA or miRNA molecule or complement thereof. While in someembodiments, nucleic acids of the invention do not have an entiresequence that is identical or complementary to a sequence of anaturally-occurring nucleic acid, such molecules may encompass all orpart of a naturally-occurring sequence or a complement thereof. It iscontemplated, however, that a synthetic nucleic acid administered to acell may subsequently be modified or altered in the cell such that itsstructure or sequence is the same as non-synthetic or naturallyoccurring nucleic acid, such as a mature miRNA sequence. For example, asynthetic nucleic acid may have a sequence that differs from thesequence of a precursor miRNA, but that sequence may be altered once ina cell to be the same as an endogenous, processed miRNA or an inhibitorthereof.

The term “isolated” means that the nucleic acid molecules of theinvention are initially separated from different (in terms of sequenceor structure) and unwanted nucleic acid molecules such that a populationof isolated nucleic acids is at least about 90% homogenous, and may beat least about 95, 96, 97, 98, 99, or 100% homogenous with respect toother polynucleotide molecules. In many embodiments of the invention, anucleic acid is isolated by virtue of it having been synthesized invitro separate from endogenous nucleic acids in a cell. It will beunderstood, however, that isolated nucleic acids may be subsequentlymixed or pooled together. In certain aspects, synthetic miRNA of theinvention are RNA or RNA analogs. miRNA inhibitors may be DNA or RNA, oranalogs thereof.

In some embodiments, there is a miRNA or a synthetic miRNA having alength of between 10 and 130 residues. The present invention concernsmiRNA or synthetic miRNA molecules that are, are at least, or are atmost 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 140, 145, 150, 160, 170, 180, 190, 200 or more residues inlength, including any integer or any range there between.

In certain embodiments, synthetic miRNA have (a) a “miRNA region” whosesequence or binding region from 5′ to 3′ is identical or complementaryto all or a segment of a mature miRNA sequence, and (b) a “complementaryregion” whose sequence from 5′ to 3′ is between 60% and 100%complementary to the miRNA sequence in (a). In certain embodiments,these synthetic miRNA are also isolated, as defined above. The term“miRNA region” or complement thereof refers to a region on the syntheticmiRNA that is at least 75, 80, 85, 90, 95, or 100% identical, includingall integers there between, to the entire sequence of a mature,naturally occurring miRNA sequence or a complement thereof. In certainembodiments, the miRNA region is or is at least 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or100% identical to the sequence of a naturally-occurring miRNA orcomplement thereof. In certain aspects, a double stranded RNA cancomprise a miR sequence that is 90 to 100% identical to sequencesdescribed herein, as described directly above, and a second nucleic acidthat is complementary to the miR sequence and is 60, 65, 70, 75, 80, 85,90, 95, or 100% identical, including all integers there between, to themiR sequence.

The term “complementary region” or “complement” refers to a region of anucleic acid or mimetic that is or is at least 60% complementary to themature, naturally occurring miRNA sequence. The complementary region canbe at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7,99.8, 99.9 or 100% complementary including all values and ranges therebetween. With single polynucleotide sequences, there may be a hairpinloop structure as a result of chemical bonding between the miRNA regionand the complementary region. In other embodiments, the complementaryregion is on a different nucleic acid molecule than the miRNA region, inwhich case the complementary region is on the complementary strand andthe miRNA region is on the active strand.

In some embodiments of the invention a synthetic miRNA contains one ormore design element(s). These design elements include, but are notlimited to: (i) a replacement group for the phosphate or hydroxyl of thenucleotide at the 5′ terminus of the complementary region; (ii) one ormore sugar modifications in the first or last 1 to 6 residues of thecomplementary region; or, (iii) noncomplementarity between one or morenucleotides in the last 1 to 5 residues at the 3′ end of thecomplementary region and the corresponding nucleotides of the miRNAregion. A variety of design modifications are known in the art, seebelow.

In certain embodiments, a synthetic miRNA has a nucleotide at its 5′ endof the complementary region in which the phosphate and/or hydroxyl grouphas been replaced with another chemical group (referred to as the“replacement design”). In some cases, the phosphate group is replaced,while in others, the hydroxyl group has been replaced. In particularembodiments, the replacement group is biotin, an amine group, a loweralkylamine group, an acetyl group, 2′O-Me (2′ oxygen-methyl), DMTO(4,4′-dimethoxytrityl with oxygen), fluoroscein, a thiol, or acridine,though other replacement groups are well known to those of skill in theart and can be used as well. This design element can also be used with amiRNA inhibitor.

Additional embodiments concern a synthetic miRNA having one or moresugar modifications in the first or last 1 to 6 residues of thecomplementary region (referred to as the “sugar replacement design”). Incertain cases, there is one or more sugar modifications in the first 1,2, 3, 4, 5, 6 or more residues of the complementary region, or any rangederivable therein. In additional cases, there are one or more sugarmodifications in the last 1, 2, 3, 4, 5, 6 or more residues of thecomplementary region, or any range derivable therein, have a sugarmodification. It will be understood that the terms “first” and “last”are with respect to the order of residues from the 5′ end to the 3′ endof the region. In particular embodiments, the sugar modification is a2′O-Me modification, a 2° F. modification, a 2′H modification, a 2′aminomodification, a 4′thioribose modification or a phosphorothioatemodification on the carboxy group linked to the carbon at position 6′.In further embodiments, there are one or more sugar modifications in thefirst or last 2 to 4 residues of the complementary region or the firstor last 4 to 6 residues of the complementary region. This design elementcan also be used with a miRNA inhibitor. Thus, a miRNA inhibitor canhave this design element and/or a replacement group on the nucleotide atthe 5′ terminus, as discussed above.

In other embodiments of the invention, there is a synthetic miRNA inwhich one or more nucleotides in the last 1 to 5 residues at the 3′ endof the complementary region are not complementary to the correspondingnucleotides of the miRNA region (“noncomplementarity”) (referred to asthe “noncomplementarity design”). The noncomplementarity may be in thelast 1, 2, 3, 4, and/or 5 residues of the complementary miRNA. Incertain embodiments, there is noncomplementarity with at least 2nucleotides in the complementary region.

It is contemplated that synthetic miRNA of the invention have one ormore of the replacement, sugar modification, or noncomplementaritydesigns. In certain cases, synthetic RNA molecules have two of them,while in others these molecules have all three designs in place.

The miRNA region and the complementary region may be on the same orseparate polynucleotides. In cases in which they are contained on or inthe same polynucleotide, the miRNA molecule will be considered a singlepolynucleotide. In embodiments in which the different regions are onseparate polynucleotides, the synthetic miRNA will be considered to becomprised of two polynucleotides.

When the RNA molecule is a single polynucleotide, there can be a linkerregion between the miRNA region and the complementary region. In someembodiments, the single polynucleotide is capable of forming a hairpinloop structure as a result of bonding between the miRNA region and thecomplementary region. The linker constitutes the hairpin loop. It iscontemplated that in some embodiments, the linker region is, is atleast, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 residues in length, or any range derivabletherein. In certain embodiments, the linker is between 3 and 30 residues(inclusive) in length.

In addition to having a miRNA region and a complementary region, theremay be flanking sequences as well at either the 5′ or 3′ end of theregion. In some embodiments, there is or is at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10 nucleotides or more, or any range derivable therein,flanking one or both sides of these regions.

In some embodiments of the invention, methods and compositions involvingmiRNA may concern nucleic acids comprising miRNA nucleotide sequences.Nucleic acids may be, be at least, or be at most 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700,710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840,850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980,990, or 1000 nucleotides, or any range derivable therein, in length.Such lengths cover the lengths of processed miRNA, precursor miRNA,miRNA containing vectors, and therapeutic miRNA. In many embodiments,miRNA are 14-35 nucleotides in length. miRNA precursors are generallybetween 62 and 10 nucleotides in humans.

It is understood that some nucleic acids are derived from genomicsequences or a gene. In this respect, the term “gene” is used forsimplicity to refer to the genomic sequence encoding the precursornucleic acid or miRNA for a given miRNA or gene. However, embodiments ofthe invention may involve genomic sequences of a miRNA that are involvedin its expression, such as a promoter or other regulatory sequences.

The term “recombinant” may be used and this generally refers to amolecule that has been manipulated in vitro or that is a replicated orexpressed product of such a molecule.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (one or more strands) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompasses the terms “oligonucleotide”and “polynucleotide,” each as a subgenus of the term “nucleic acid.”

The term “miRNA” generally refers to a single-stranded molecule, but inspecific embodiments, molecules implemented in the invention will alsoencompass a region or an additional strand that is partially (between 10and 50% complementary across length of strand), substantially (greaterthan 50% but less than 100% complementary across length of strand) orfully complementary to another region of the same single-strandedmolecule or to another nucleic acid. Thus, miRNA nucleic acids mayencompass a molecule that comprises one or more complementary orself-complementary strand(s) or “complement(s)” of a particularsequence. For example, precursor miRNA may have a self-complementaryregion, which is up to 100% complementary. miRNA probes or nucleic acidsof the invention can include, can be or can be at least 60, 65, 70, 75,80, 85, 90, 95, 96, 97, 98, 99 or 100% complementary to their target.

It is understood that a “synthetic nucleic acid” of the invention meansthat the nucleic acid does not have all or part of a chemical structureor sequence of a naturally occurring nucleic acid or was made by man andnot a biologic cell or organism. Consequently, it will be understoodthat the term “synthetic miRNA” refers to a “synthetic nucleic acid”that functions in a cell or under physiological conditions as anaturally occurring miRNA.

While embodiments of the invention may involve synthetic miRNAs orsynthetic nucleic acids, in some embodiments of the invention, thenucleic acid molecule(s) need not be “synthetic.” In certainembodiments, a non-synthetic nucleic acid or miRNA employed in methodsand compositions of the invention may have the entire sequence andstructure of a naturally occurring mRNA or miRNA precursor or the maturemRNA or miRNA. For example, non-synthetic miRNAs used in methods andcompositions of the invention may not have one or more modifiednucleotides or nucleotide analogs. In these embodiments, thenon-synthetic miRNA may or may not be recombinantly produced. Inparticular embodiments, the nucleic acid in methods and/or compositionsof the invention is specifically a synthetic miRNA and not anon-synthetic miRNA (that is, not a miRNA that qualifies as“synthetic”); though in other embodiments, the invention specificallyinvolves a non-synthetic miRNA and not a synthetic miRNA. Anyembodiments discussed with respect to the use of synthetic miRNAs can beapplied with respect to non-synthetic miRNAs, and vice versa.

It will be understood that the term “naturally occurring” refers tosomething found in an organism without any intervention by a person; itcould refer to a naturally-occurring wildtype or mutant molecule. Insome embodiments a synthetic miRNA molecule does not have the sequenceof a naturally occurring miRNA molecule. In other embodiments, asynthetic miRNA molecule may have the sequence of a naturally occurringmiRNA molecule, but the chemical structure of the molecule, particularlyin the part unrelated specifically to the precise sequence (non-sequencechemical structure) differs from chemical structure of the naturallyoccurring miRNA molecule with that sequence. In some cases, thesynthetic miRNA has both a sequence and non-sequence chemical structurethat are not found in a naturally-occurring miRNA. Moreover, thesequence of the synthetic molecules will identify which miRNA iseffectively being provided; the endogenous miRNA will be referred to asthe “corresponding miRNA.” Corresponding miRNA sequences that can beused in the context of the invention include, but are not limited to,all or a portion of those sequences in the SEQ IDs provided herein, aswell as any other miRNA sequence, miRNA precursor sequence, or anysequence complementary thereof. In some embodiments, the sequence is oris derived from or contains all or part of a sequence identified hereinto target a particular miRNA (or set of miRNAs) that can be used withthat sequence. Any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or any numberor range of sequences there between may be selected to the exclusion ofall non-selected sequences.

1. Nucleobase, Nucleoside, Nucleotide, and Modified Nucleotides

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in a manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, carboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moiety. Preferred alkyl (e.g., alkyl, carboxyalkyl, etc.)moieties comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like. Other examplesare well known to those of skill in the art.

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring. Different types ofcovalent attachment(s) of a nucleobase to a nucleobase linker moiety areknown in the art (Kornberg and Baker, 1992).

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. RNAwith nucleic acid analogs may also be labeled according to methods ofthe invention. As used herein a “derivative” refers to a chemicallymodified or altered form of a naturally occurring molecule, while theterms “mimic” or “analog” refer to a molecule that may or may notstructurally resemble a naturally occurring molecule or moiety, butpossesses similar functions. As used herein, a “moiety” generally refersto a smaller chemical or molecular component of a larger chemical ormolecular structure. Nucleobase, nucleoside and nucleotide analogs orderivatives are well known in the art, and have been described (see forexample, Scheit, 1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids include those in: U.S. Pat. Nos. 5,681,947, 5,652,099 and5,763,167, 5,614,617, 5,670,663, 5,872,232, 5,859,221, 5,446,137,5,886,165, 5,714,606, 5,672,697, 5,466,786, 5,792,847, 5,223,618,5,470,967, 5,378,825, 5,777,092, 5,623,070, 5,610,289, 5,602,240,5,858,988, 5,214,136, 5,700,922, 5,708,154, 5,728,525, 5,637,683,6,251,666, 5,480,980, and 5,728,525, each of which is incorporatedherein by reference in its entirety.

Labeling methods and kits of the invention specifically contemplate theuse of nucleotides that are both modified for attachment of a label andcan be incorporated into a miRNA molecule. Such nucleotides includethose that can be labeled with a dye, including a fluorescent dye, orwith a molecule such as biotin. Labeled nucleotides are readilyavailable; they can be acquired commercially or they can be synthesizedby reactions known to those of skill in the art.

Modified nucleotides for use in the invention are not naturallyoccurring nucleotides, but instead, refer to prepared nucleotides thathave a reactive moiety on them. Specific reactive functionalities ofinterest include: amino, sulfhydryl, sulfoxyl, aminosulfhydryl, azido,epoxide, isothiocyanate, isocyanate, anhydride, monochlorotriazine,dichlorotriazine, mono- or dihalogen substituted pyridine, mono- ordisubstituted diazine, maleimide, epoxide, aziridine, sulfonyl halide,acid halide, alkyl halide, aryl halide, alkylsulfonate,N-hydroxysuccinimide ester, imido ester, hydrazine, azidonitrophenyl,azide, 3-(2-pyridyl dithio)-propionamide, glyoxal, aldehyde, iodoacetyl,cyanomethyl ester, p-nitrophenyl ester, o-nitrophenyl ester,hydroxypyridine ester, carbonyl imidazole, and the other such chemicalgroups. In some embodiments, the reactive functionality may be bondeddirectly to a nucleotide, or it may be bonded to the nucleotide througha linking group. The functional moiety and any linker cannotsubstantially impair the ability of the nucleotide to be added to themiRNA or to be labeled. Representative linking groups include carboncontaining linking groups, typically ranging from about 2 to 18, usuallyfrom about 2 to 8 carbon atoms, where the carbon containing linkinggroups may or may not include one or more heteroatoms, e.g. S, O, Netc., and may or may not include one or more sites of unsaturation. Ofparticular interest in many embodiments is alkyl linking groups,typically lower alkyl linking groups of 1 to 16, usually 1 to 4 carbonatoms, where the linking groups may include one or more sites ofunsaturation. The functionalized nucleotides (or primers) used in theabove methods of functionalized target generation may be fabricatedusing known protocols or purchased from commercial vendors, e.g., Sigma,Roche, Ambion, Biosearch Technologies and NEN. Functional groups may beprepared according to ways known to those of skill in the art, includingthe representative information found in U.S. Pat. Nos. 4,404,289;4,405,711; 4,337,063 and 5,268,486, and U.K. Patent 1,529,202, which areall incorporated by reference.

Amine-modified nucleotides are used in several embodiments of theinvention. The amine-modified nucleotide is a nucleotide that has areactive amine group for attachment of the label. It is contemplatedthat any ribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T,or C) can be modified for labeling. Examples include, but are notlimited to, the following modified ribo- and deoxyribo-nucleotides:5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP,N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP;N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP;5-propargylamino-CTP, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP;8-[(4-amino)butyl]-amino-dATP and 8-[(6-amino)butyl]-amino-dATP;N6-(4-amino)butyl-dATP, N6-(6-amino)butyl-dATP,N4-[2,2-oxy-bis-(ethylamine)]-dCTP; N6-(6-Amino)hexyl-dATP;8-[(6-Amino)hexyl]-amino-dATP; 5-propargylamino-dCTP, and5-propargylamino-dUTP. Such nucleotides can be prepared according tomethods known to those of skill in the art. Moreover, a person ofordinary skill in the art could prepare other nucleotide entities withthe same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP,dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoallyl)-UTP.

B. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction, or biological production. It is specifically contemplatedthat miRNA probes of the invention are chemically synthesized.

In some embodiments of the invention, miRNAs are recovered or isolatedfrom a biological sample. The miRNA may be recombinant or it may benatural or endogenous to the cell (produced from the cell's genome). Itis contemplated that a biological sample may be treated in a way so asto enhance the recovery of small RNA molecules such as miRNA. U.S.patent application Ser. No. 10/667,126 describes such methods and it isspecifically incorporated by reference herein. Generally, methodsinvolve lysing cells with a solution having guanidinium and a detergent.

Alternatively, nucleic acid synthesis is performed according to standardmethods. See, for example, Itakura and Riggs (1980) and U.S. Pat. Nos.4,704,362, 5,221,619, and 5,583,013, each of which is incorporatedherein by reference. Non-limiting examples of a synthetic nucleic acid(e.g., a synthetic oligonucleotide), include a nucleic acid made by invitro chemical synthesis using phosphotriester, phosphite, orphosphoramidite chemistry and solid phase techniques such as describedin EP 266,032, incorporated herein by reference, or via deoxynucleosideH-phosphonate intermediates as described by Froehler et al., 1986 andU.S. Pat. No. 5,705,629, each incorporated herein by reference. Variousdifferent mechanisms of oligonucleotide synthesis have been disclosed infor example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. Nos. 4,683,202 and 4,682,195, each incorporatedherein by reference), or the synthesis of an oligonucleotide describedin U.S. Pat. No. 5,645,897, incorporated herein by reference. See alsoSambrook et al., 2001, incorporated herein by reference).

Oligonucleotide synthesis is well known to those of skill in the art.Various different mechanisms of oligonucleotide synthesis have beendisclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

Recombinant methods for producing nucleic acids in a cell are well knownto those of skill in the art. These include the use of vectors (viraland non-viral), plasmids, cosmids, and other vehicles for delivering anucleic acid to a cell, which may be the target cell (e.g., a cancercell) or simply a host cell (to produce large quantities of the desiredRNA molecule). Alternatively, such vehicles can be used in the contextof a cell free system so long as the reagents for generating the RNAmolecule are present. Such methods include those described in Sambrook,2003, Sambrook, 2001 and Sambrook, 1989, which are hereby incorporatedby reference.

C. Isolation of Nucleic Acids

Nucleic acids may be isolated using techniques well known to those ofskill in the art, though in particular embodiments, methods forisolating small nucleic acid molecules, and/or isolating RNA moleculescan be employed. Chromatography is a process often used to separate orisolate nucleic acids from protein or from other nucleic acids. Suchmethods can involve electrophoresis with a gel matrix, filter columns,alcohol precipitation, and/or other chromatography. If miRNA from cellsis to be used or evaluated, methods generally involve lysing the cellswith a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent(e.g., N-lauroyl sarcosine) prior to implementing processes forisolating particular populations of RNA.

In particular methods for separating miRNA from other nucleic acids, agel matrix is prepared using polyacrylamide, though agarose can also beused. The gels may be graded by concentration or they may be uniform.Plates or tubing can be used to hold the gel matrix for electrophoresis.Usually one-dimensional electrophoresis is employed for the separationof nucleic acids. Plates are used to prepare a slab gel, while thetubing (glass or rubber, typically) can be used to prepare a tube gel.The phrase “tube electrophoresis” refers to the use of a tube or tubing,instead of plates, to form the gel. Materials for implementing tubeelectrophoresis can be readily prepared by a person of skill in the artor purchased, such as from C.B.S. Scientific Co., Inc. or Scie-Plas.

Methods may involve the use of organic solvents and/or alcohol toisolate nucleic acids, particularly miRNA used in methods andcompositions of the invention. Some embodiments are described in U.S.patent application Ser. No. 10/667,126, which is hereby incorporated byreference. Generally, this disclosure provides methods for efficientlyisolating small RNA molecules from cells comprising: adding an alcoholsolution to a cell lysate and applying the alcohol/lysate mixture to asolid support before eluting the RNA molecules from the solid support.In some embodiments, the amount of alcohol added to a cell lysateachieves an alcohol concentration of about 55% to 60%. While differentalcohols can be employed, ethanol works well. A solid support may be anystructure, and it includes beads, filters, and columns, which mayinclude a mineral or polymer support with electronegative groups. Aglass fiber filter or column has worked particularly well for suchisolation procedures.

In specific embodiments, miRNA isolation processes include: (a) lysingcells in the sample with a lysing solution comprising guanidinium,wherein a lysate with a concentration of at least about 1 M guanidiniumis produced; (b) extracting miRNA molecules from the lysate with anextraction solution comprising phenol; (c) adding to the lysate analcohol solution for forming a lysate/alcohol mixture, wherein theconcentration of alcohol in the mixture is between about 35% to about70%; (d) applying the lysate/alcohol mixture to a solid support; (e)eluting the miRNA molecules from the solid support with an ionicsolution; and, (f) capturing the miRNA molecules. Typically the sampleis dried and resuspended in a liquid and volume appropriate forsubsequent manipulation.

IV. PHARMACEUTICAL FORMULATIONS AND DELIVERY

Methods of the present invention include the delivery of an effectiveamount of a miRNA or an expression construct encoding the same. An“effective amount” of the pharmaceutical composition, generally, isdefined as that amount sufficient to detectably achieve a desiredresult, for example, to ameliorate, reduce, minimize or limit the extentof a disease or its symptoms. Other more rigorous definitions may apply,including elimination, eradication or cure of disease.

A. Administration

In certain embodiments, it is desired to inhibit vascularization,stimulate vascularization, kill cells, inhibit cell growth, inhibitmetastasis, decrease tumor or tissue size, and/or reverse or reduce themalignant or disease phenotype of cells. The routes of administrationwill vary, naturally, with the location and nature of the lesion or siteto be targeted, and include, e.g., intradermal, subcutaneous, regional,parenteral, intravenous, intramuscular, intranasal, systemic, and oraladministration and formulation. Direct injection, local injection, orinjection into vasculature at a target site is specifically contemplatedfor target areas. Local, regional, or systemic administration also maybe appropriate.

Multiple injections delivered as a single dose comprise about 0.1 toabout 0.5 ml volumes. Compositions of the invention may be administeredin multiple injections to a tumor or a targeted site. In certainaspects, injections may be spaced at approximately 1 cm intervals.

In the case of surgical intervention, the present invention may be usedpreoperatively, to render an inoperable target area subject toresection. Alternatively, the present invention may be used at the timeof surgery, and/or thereafter, to treat residual or recurring disease.For example, a resected tumor bed may be injected or perfused with aformulation comprising a miRNA or combinations thereof. Administrationmay be continued post-resection, for example, by leaving a catheterimplanted at the site of the surgery. Periodic post-surgical treatmentalso is envisioned. Continuous perfusion of an expression construct or aviral construct also is contemplated.

Continuous administration also may be applied where appropriate, forexample, where a tumor or other undesired affected area is excised andthe tumor bed or targeted site is treated to eliminate residual,microscopic disease. Delivery via syringe or catherization iscontemplated. Such continuous perfusion may take place for a period fromabout 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about 12-24hours, to about 1-2 days, to about 1-2 wk or longer following theinitiation of treatment. Generally, the dose of the therapeuticcomposition via continuous perfusion will be equivalent to that given bya single or multiple injections, adjusted over a period of time duringwhich the perfusion occurs.

Treatment regimens may vary as well and often depend on lesion type,tumor type, location, immune condition, target site, diseaseprogression, and health and age of the patient. Certain tumor types willrequire more aggressive treatment. The clinician will be best suited tomake such decisions based on the known efficacy and toxicity (if any) ofthe therapeutic formulations.

In certain embodiments, the tumor or affected area being treated maynot, at least initially, be resectable or operable. Treatments withcompositions of the invention may increase the resectability of thetumor or target site due to shrinkage at the margins or by eliminationof certain particularly invasive portions. Following treatments,resection may be possible. Additional treatments subsequent to resectionmay serve to eliminate microscopic residual disease at the tumor ortargeted site.

Treatments may include various “unit doses.” A unit dose is defined ascontaining a predetermined quantity of a therapeutic composition(s). Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. With respect to a viral component ofthe present invention, a unit dose may conveniently be described interms of μg or mg of miRNA or miRNA mimetic. Alternatively, the amountspecified may be the amount administered as the average daily, averageweekly, or average monthly dose.

miRNA can be administered to the patient in a dose or doses of about orof at least about 0.005, 0.05, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320,330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600,610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 μg, ng, ormg, or more, or any range derivable therein. Alternatively, the amountspecified may be the amount administered as the average daily, averageweekly, or average monthly dose, or it may be expressed in terms ofmg/kg, where kg refers to the weight of the patient and the mg isspecified above. In other embodiments, the amount specified is anynumber discussed above but expressed as mg/m² (with respect to tumorsize or patient surface area).

B. Injectable Compositions and Formulations

In some embodiments, the method for the delivery of a miRNA or anexpression construct encoding such or combinations thereof is viasystemic administration. However, the pharmaceutical compositionsdisclosed herein may also be administered topically, parenterally,subcutaneously, directly, intratracheally, intravenously, intradermally,intramuscularly, or even intraperitoneally as described in U.S. Pat.Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporatedherein by reference in its entirety).

Injection of nucleic acids may be delivered by syringe or any othermethod used for injection of a solution, as long as the nucleic acid andany associated components can pass through the particular gauge ofneedle required for injection. A syringe system has also been describedfor use in gene therapy that permits multiple injections ofpredetermined quantities of a solution precisely at any depth (U.S. Pat.No. 5,846,225).

Solutions of the active compounds are typically sterile and must befluid to the extent that easy syringability exists. It must besufficiently stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.

In certain formulations, a water-based formulation is employed while inothers, it may be lipid-based. In particular embodiments of theinvention, a composition comprising a tumor suppressor protein or anucleic acid encoding the same is in a water-based formulation. In otherembodiments, the formulation is lipid based.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intratumoral, intralesional, andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. For example, one dosage may bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety, and purity standards as required by FDA Office ofBiologics standards.

As used herein, a “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human.

The nucleic acid(s) are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective. The quantity to be administered depends on the subject to betreated, including, e.g., the aggressiveness of the disease or cancer,the size of any tumor(s) or lesions, the previous or other courses oftreatment. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner. Suitableregimes for initial administration and subsequent administration arealso variable, but are typified by an initial administration followed byother administrations. Such administration may be systemic, as a singledose, continuous over a period of time spanning 10, 20, 30, 40, 50, 60minutes, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24 or more hours, and/or 1, 2, 3, 4, 5, 6,7, days or more. Moreover, administration may be through a time releaseor sustained release mechanism, implemented by formulation and/or modeof administration.

C. Combination Treatments

In certain embodiments, the compositions and methods of the presentinvention involve a miRNA, or miRNA inhibitor, or expression constructencoding such. These miRNA compositions can be used in combination witha second therapy to enhance the effect of the miRNA therapy, or increasethe therapeutic effect of another therapy being employed. Thesecompositions would be provided in a combined amount effective to achievethe desired effect, such as inhibiting or stimulating vascularization,killing of a cancer cell and/or inhibition of cellularhyperproliferation. This process may involve contacting the cells withthe miRNA or second therapy at the same or different time. This may beachieved by contacting the cell with one or more compositions orpharmacological formulation that includes or more of the agents, or bycontacting the cell with two or more distinct compositions orformulations, wherein one composition provides (1) miRNA; and/or (2) asecond therapy. A second composition or method may be administered thatincludes an antiangiogenic or pro-angiogenic therapy, chemotherapy,radiotherapy, surgical therapy, immunotherapy, or gene therapy.

It is contemplated that one may provide a patient with the miRNA therapyand the second therapy within about 12-24 h of each other and, morepreferably, within about 6-12 h of each other. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In certain embodiments, a course of treatment will last 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more. It iscontemplated that one agent may be given on day 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any combination thereof,and another agent is given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, and/or 90, or any combination thereof. Within asingle day (24-hour period), the patient may be given one or multipleadministrations of the agent(s). Moreover, after a course of treatment,it is contemplated that there is a period of time at which no treatmentis administered. This time period may last 1, 2, 3, 4, 5, 6, 7 days,and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12months or more, depending on the condition of the patient, such as theirprognosis, strength, health, etc.

Various combinations may be employed, for example miRNA therapy is “A”and a second therapy is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of any compound or therapy of the present invention to apatient will follow general protocols for the administration of suchcompounds, taking into account the toxicity, if any, of the vector orany protein or other agent. Therefore, in some embodiments there is astep of monitoring toxicity that is attributable to combination therapy.It is expected that the treatment cycles would be repeated as necessary.It also is contemplated that various standard therapies, as well assurgical intervention, may be applied in combination with the describedtherapy. Cancer therapeutics can involve the application of multipletargeted therapies that include Tarceva, Iressa, Hierceptin, and othertargeted therapeutics along with radiation and chemotherapies.

In specific aspects, it is contemplated that a second therapy, such aschemotherapy, radiotherapy, immunotherapy, surgical therapy or othergene therapy, is employed in combination with the miRNA therapy, asdescribed herein.

1. Antiangiogenic Therapy

Antiangiogenic therapy includes, but is not limited to Macugen,Visudyne, and Photodynamic Therapy (PDT) for macular degeneration andother diseases of the eye.

Angiogenesis inhibitors which inhibit angiogenesis in treated tissuescan be used in the compositions and methods of the invention.Angiogenesis inhibitors include α_(v) antagonist, and in particular aα_(v)β₃ antagonist. An angiogenesis inhibiting (anti-angiogenesis)α_(v)β₃ antagonist can be a peptide, a RGD-containing peptide, ananti-α_(v)β₃₃ antibody, an anti-α_(v)β₃ receptor antibody, or an α_(v)β₃mimetic. Exemplary antiangiogenic substances are described in theteachings of U.S. Pat. Nos. 5,753,230, 5,766,591, and U.S. Patentpublications 20080039384, 20080014196, 20080096795, 20080090750,International Publication No. WO 97/45137, the disclosures of which arespecifically incorporated herein by reference.

Other compounds have been identified on the basis of the ability for thecompound to inhibit angiogenesis and include but are not limited toprostate specific antigen (PSA); soluble VEGFR-1 and NRP-1; Angiopoietin2; TSP-1 and TSP-2; angiostatin and related molecules; endostatin;vasostatin; calreticulin; platelet factor-4; TIMP and CDAI; Meth-1 andMeth-2; IFN-α, -β and -γ; CXCL10; IL-4, -12 and -18; prothrombin(kringle domain-2); antithrombin III fragment; prolactin; VEGI; SPARC;osteopontin; maspin; canstatin; proliferin-related protein; restin;bevacizumab; carboxyamidotriazole; TNP-470; CM10; suramin; SU5416;thrombospondin; VEGFR antagonists; angiostatic steroids+heparin;Cartilage-Derived Angiogenesis Inhibitory Factor; matrixmetalloproteinase inhibitors; 2-methoxyestradiol; tecogalan;thrombospondin; prolactin; αvβ3 inhibitors; and linomide.

2. Chemotherapy

A wide variety of chemotherapeutic agents may be used in accordance withthe present invention. The term “chemotherapy” refers to the use ofdrugs to treat cancer or other hyperproliferative diseases. A“chemotherapeutic agent” is used to connote a compound or compositionthat is administered in the treatment of cancer or otherhyperproliferative disease. These agents or drugs are categorized bytheir mode of activity within a cell, for example, whether and at whatstage they affect the cell cycle. Alternatively, an agent may becharacterized based on its ability to directly cross-link DNA, tointercalate into DNA, or to induce chromosomal and mitotic aberrationsby affecting nucleic acid synthesis. Most chemotherapeutic agents fallinto the following categories: alkylating agents, antimetabolites,antitumor antibiotics, mitotic inhibitors, and nitrosoureas.

a. Alkylating Agents

Alkylating agents are drugs that directly interact with genomic DNA toprevent the cancer cell from proliferating. This category ofchemotherapeutic drugs represents agents that affect all phases of thecell cycle, that is, they are not phase-specific. Alkylating agents canbe implemented to treat chronic leukemia, non-Hodgkin's lymphoma,Hodgkin's disease, multiple myeloma, and particular cancers of thebreast, lung, and ovary. They include: busulfan, chlorambucil,cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide,mechlorethamine (mustargen), and melphalan. Troglitazaone can be used totreat cancer in combination with any one or more of these alkylatingagents.

b. Antimetabolites

Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents,they specifically influence the cell cycle during S phase. They havebeen used to combat chronic leukemias in addition to tumors of breast,ovary and the gastrointestinal tract. Antimetabolites include5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, andmethotrexate.

5-Fluorouracil (5-FU) has the chemical name of5-fluoro-2,4(1H,3H)-pyrimidinedione. Its mechanism of action is thoughtto be by blocking the methylation reaction of deoxyuridylic acid tothymidylic acid. Thus, 5-FU interferes with the synthesis ofdeoxyribonucleic acid (DNA) and to a lesser extent inhibits theformation of ribonucleic acid (RNA). Since DNA and RNA are essential forcell division and proliferation, it is thought that the effect of 5-FUis to create a thymidine deficiency leading to cell death. Thus, theeffect of 5-FU is found in cells that rapidly divide, a characteristicof metastatic cancers.

c. Antitumor Antibiotics

Antitumor antibiotics have both antimicrobial and cytotoxic activity.These drugs also interfere with DNA by chemically inhibiting enzymes andmitosis or altering cellular membranes. These agents are not phasespecific so they work in all phases of the cell cycle. Thus, they arewidely used for a variety of cancers. Examples of antitumor antibioticsinclude bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin),and idarubicin, some of which are discussed in more detail below. Widelyused in clinical setting for the treatment of neoplasms, these compoundsare administered through bolus injections intravenously at doses rangingfrom 25-75 mg/m² at 21 day intervals for adriamycin, to 35-100 mg/m² foretoposide intravenously or orally.

d. Mitotic Inhibitors

Mitotic inhibitors include plant alkaloids and other natural agents thatcan inhibit either protein synthesis required for cell division ormitosis. They operate during a specific phase during the cell cycle.Mitotic inhibitors comprise docetaxel, etoposide (VP16), paclitaxel,taxol, taxotere, vinblastine, vincristine, and vinorelbine.

e. Nitrosureas

Nitrosureas, like alkylating agents, inhibit DNA repair proteins. Theyare used to treat non-Hodgkin's lymphomas, multiple myeloma, malignantmelanoma, in addition to brain tumors. Examples include carmustine andlomustine.

3. Radiotherapy

Radiotherapy, also called radiation therapy, is the treatment of cancerand other diseases with ionizing radiation. Ionizing radiation depositsenergy that injures or destroys cells in the area being treated bydamaging their genetic material, making it impossible for these cells tocontinue to grow. Although radiation damages both cancer cells andnormal cells, the latter are able to repair and function properly.Radiotherapy may be used to treat localized solid tumors, such ascancers of the skin, tongue, larynx, brain, breast, or cervix. It canalso be used to treat leukemia and lymphoma (cancers of theblood-forming cells and lymphatic system, respectively).

Radiation therapy used according to the present invention may include,but is not limited to, the use of γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves, proton beamirradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287) and UV-irradiation.It is most likely that all of these factors affect a broad range ofdamage on DNA, on the precursors of DNA, on the replication and repairof DNA, and on the assembly and maintenance of chromosomes. Dosageranges for X-rays range from daily doses of 50 to 200 roentgens forprolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000roentgens. Dosage ranges for radioisotopes vary widely, and depend onthe half-life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells. Radiotherapy maycomprise the use of radiolabeled antibodies to deliver doses ofradiation directly to the cancer site (radioimmunotherapy). Onceinjected into the body, the antibodies actively seek out the cancercells, which are destroyed by the cell-killing (cytotoxic) action of theradiation. This approach can minimize the risk of radiation damage tohealthy cells.

4. Immunotherapy

In the context of cancer treatment, immunotherapeutics, generally, relyon the use of immune effector cells and molecules to target and destroycancer cells. Trastuzumab (Herceptin™) is such an example. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a target cell. The antibody alone may serve as aneffector of therapy or it may recruit other cells to actually affectcell killing. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a target cell target. Variouseffector cells include cytotoxic T cells and NK cells.

In one aspect of immunotherapy, the tumor or disease cell must bear somemarker that is amenable to targeting, i.e., is not present on themajority of other cells. Many tumor markers exist and any of these maybe suitable for targeting in the context of the present invention.Common tumor markers include carcinoembryonic antigen, prostate specificantigen, urinary tumor associated antigen, fetal antigen, tyrosinase(p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP,estrogen receptor, laminin receptor, erb B and p155. An alternativeaspect of immunotherapy is to combine anticancer effects with immunestimulatory effects. Immune stimulating molecules also exist including:cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, and chemokinessuch as MIP-1, MCP-1, IL-8 and growth factors such as FLT3 ligand.Antibodies against any of these compounds can be used to target theanti-cancer agents discussed herein.

Examples of immunotherapies currently under investigation or in use areimmune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum,dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998),cytokine therapy, e.g., interferons α, β and γ; IL-1, GM-CSF and TNF(Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998)gene therapy e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Wardand Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) andmonoclonal antibodies e.g., anti-ganglioside GM2, anti-HER-2, anti-p185;Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311).A non-limiting list of several known anti-cancer immunotherapeuticagents and their targets includes, but is not limited to (Generic Name(Target)) Cetuximab (EGFR), Panitumumab (EGFR), Trastuzumab (erbB2receptor), Bevacizumab (VEGF), Alemtuzumab (CD52), Gemtuzumab ozogamicin(CD33), Rituximab (CD20), Tositumomab (CD20), Matuzumab (EGFR),Ibritumomab tiuxetan (CD20), Tositumomab (CD20), HuPAM4 (MUC1),MORAb-009 (Mesothelin), G250 (carbonic anhydrase IX), mAb 8H9 (8H9antigen), M195 (CD33), Ipilimumab (CTLA4), HuLuc63 (CS1), Alemtuzumab(CD53), Epratuzumab (CD22), BC8 (CD45), HuJ591 (Prostate specificmembrane antigen), hA20 (CD20), Lexatumumab (TRAIL receptor-2),Pertuzumab (HER-2 receptor), Mik-beta-1 (IL-2R), RAV12 (RAAG12), SGN-30(CD30), AME-133v (CD20), HeFi-1 (CD30), BMS-663513 (CD137), Volociximab(anti-Q5 μl integrin), GC1008 (TGFβ), HCD122 (CD40), Siplizumab (CD2),MORAb-003 (Folate receptor alpha), CNTO 328 (IL-6), MDX-060 (CD30),Ofatumumab (CD20), or SGN-33 (CD33). It is contemplated that one or moreof these therapies may be employed with the miRNA therapies describedherein.

A number of different approaches for passive immunotherapy of cancerexist. They may be broadly categorized into the following: injection ofantibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow.

5. Gene Therapy

In yet another embodiment, a combination treatment involves gene therapyin which a therapeutic polynucleotide is administered before, after, orat the same time as one or more therapeutic miRNA. Delivery of atherapeutic polypeptide or encoding nucleic acid in conjunction with amiRNA may have a combined therapeutic effect on target tissues. Avariety of proteins are encompassed within the invention, some of whichare described below. Various genes that may be targeted for gene therapyof some form in combination with the present invention include, but arenot limited to inducers of cellular proliferation, inhibitors ofcellular proliferation, regulators of programmed cell death, cytokinesand other therapeutic nucleic acids or nucleic acid that encodetherapeutic proteins.

The tumor suppressor oncogenes function to inhibit excessive cellularproliferation. The inactivation of these genes destroys their inhibitoryactivity, resulting in unregulated proliferation. The tumor suppressors(e.g., therapeutic polypeptides) p53, FHIT, p16 and C-CAM can beemployed.

Other genes that may be employed according to the present inventioninclude Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL,MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions,anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu,raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes involved inangiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or theirreceptors) and MCC.

6. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue.

Upon excision of part of all of cancerous cells, tissue, or tumor, acavity may be formed in the body. Treatment may be accomplished byperfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

Also, as described herein, inventive therapies can be used inconjunction with surgeries to the eye.

7. Other Agents

It is contemplated that other agents may be used in combination with thepresent invention to improve the therapeutic efficacy of treatment.These additional agents include immunomodulatory agents, agents thataffect the upregulation of cell surface receptors and GAP junctions,cytostatic and differentiation agents, inhibitors of cell adhesion,agents that increase the sensitivity of the hyperproliferative cells toapoptotic inducers, or other biological agents. Immunomodulatory agentsinclude tumor necrosis factor; interferon alpha, beta, and gamma; IL-2and other cytokines; F42K and other cytokine analogs; or MIP-1,MIP-1beta, MCP-1, RANTES, and other chemokines. It is furthercontemplated that the upregulation of cell surface receptors or theirligands such as Fas/Fas ligand, DR4 or DR5/TRAIL (Apo-2 ligand) wouldpotentiate the apoptotic inducing abilities of the present invention byestablishment of an autocrine or paracrine effect on hyperproliferativecells. Increases intercellular signaling by elevating the number of GAPjunctions would increase the anti-hyperproliferative effects on theneighboring hyperproliferative cell population. In other embodiments,cytostatic or differentiation agents can be used in combination with thepresent invention to improve the anti-hyperproliferative efficacy of thetreatments. Inhibitors of cell adhesion are contemplated to improve theefficacy of the present invention. Examples of cell adhesion inhibitorsare focal adhesion kinase (FAKs) inhibitors and Lovastatin. It isfurther contemplated that other agents that increase the sensitivity ofa hyperproliferative cell to apoptosis, such as the antibody c225, couldbe used in combination with the present invention to improve thetreatment efficacy.

There have been many advances in the therapy of cancer following theintroduction of cytotoxic chemotherapeutic drugs. However, one of theconsequences of chemotherapy is the development/acquisition ofdrug-resistant phenotypes and the development of multiple drugresistance. The development of drug resistance remains a major obstaclein the treatment of such tumors and therefore, there is an obvious needfor alternative approaches such as gene therapy.

Another form of therapy for use in conjunction with chemotherapy,radiation therapy or biological therapy includes hyperthermia, which isa procedure in which a patient's tissue is exposed to high temperatures(up to 106° F.). External or internal heating devices may be involved inthe application of local, regional, or whole-body hyperthermia. Localhyperthermia involves the application of heat to a small area, such as atumor. Heat may be generated externally with high-frequency wavestargeting a tumor from a device outside the body. Internal heat mayinvolve a sterile probe, including thin, heated wires or hollow tubesfilled with warm water, implanted microwave antennae, or radiofrequencyelectrodes.

A patient's organ or a limb is heated for regional therapy, which isaccomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

Hormonal therapy may also be used in conjunction with the presentinvention or in combination with any other cancer therapy previouslydescribed. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

This application incorporates U.S. application Ser. No. 11/349,727 filedon Feb. 8, 2006 claiming priority to U.S. Provisional Application Ser.No. 60/650,807 filed Feb. 8, 2005 herein by references in its entirety.

V. EVALUATION OF MIRNA LEVELS

It is contemplated that a number of assays could be employed to analyzemiRNAs, their activities, and their effects. Such assays include, butare not limited to, array hybridization, solution hybridization, nucleicacid amplification, polymerase chain reaction, quantitative PCR, RT-PCR,in situ hybridization, Northern hybridization, hybridization protectionassay (HPA) (GenProbe), branched DNA (bDNA) assay (Chiron), rollingcircle amplification (RCA), single molecule hybridization detection (USGenomics), Invader assay (ThirdWave Technologies), and/or Oligo LigationAssay (OLA), hybridization, and array analysis. U.S. patent applicationSer. Nos. 11/141,707, filed May 31, 2005; 11/857,948, filed Sep. 19,2007; 11/273,640, filed Nov. 14, 2005 and provisional patent application60/869,295, filed Dec. 8, 2006 are incorporated by reference in theirentirety.

A. Sample Preparation

While endogenous miRNA is contemplated for use with compositions andmethods of the invention, recombinant or synthetic miRNA—includingnucleic acids that are complementary or identical to endogenous miRNA orprecursor miRNA—can also be handled and analyzed as described herein.Samples may be biological samples, in which case, they can be fromlavage, biopsy, fine needle aspirates, exfoliates, blood, sputum,tissue, organs, semen, saliva, tears, urine, cerebrospinal fluid, bodyfluids, hair follicles, skin, or any sample containing or constitutingbiological cells. In certain embodiments, samples may be, but are notlimited to, fresh, frozen, fixed, formalin-fixed, preserved, RNAlater-preserved, paraffin-embedded, or formalin-fixed andparaffin-embedded. Alternatively, the sample may not be a biologicalsample, but be a chemical mixture, such as a cell-free reaction mixture(which may contain one or more biological enzymes).

B. Differential Expression Analyses

Methods of the invention can be used to detect differences in miRNAexpression or levels between two samples, or a sample and a reference(e.g., a tissue reference or a digital reference representative of anon-cancerous state). Specifically contemplated applications includeidentifying and/or quantifying differences between miRNA from a samplethat is normal and from a sample that is not normal, between a cancerouscondition and a non-cancerous condition, between two differently treatedsamples (e.g., a pretreatment versus a posttreatment sample) or betweensamples having differing prognosis. Also, miRNA may be compared betweena sample believed to be susceptible to a particular therapy or diseaseand one believed to be not susceptible or resistant to that therapy ordisease. A sample that is not normal is one exhibiting phenotypictrait(s) of a disease or condition or one believed to be not normal withrespect to that disease or condition. It may be compared to a celland/or tissue that is normal with respect to that disease or condition.Phenotypic traits include symptoms of a disease or condition of which acomponent is or may or may not be genetic or caused by a neovascularand/or angiogenic condition. It is specifically contemplated that theinvention can be used to evaluate differences between stages of adisease.

Phenotypic traits also include characteristics such as longevity,morbidity, susceptibility or receptivity to particular drugs ortherapeutic treatments (drug efficacy), and risk of drug toxicity.

In certain embodiments, miRNA profiles may be generated to evaluate andcorrelate those profiles with pharmacokinetics. For example, miRNAprofiles may be created and evaluated for patient samples prior to thepatient's being treated or during treatment to determine if there aremiRNAs whose expression correlates with the outcome of treatment.Identification of differential miRNAs can lead to a diagnostic assaythat can be used to evaluate samples to determine what drug regimen thepatient should be provided. In addition, it can be used to identify orselect patients suitable for a particular clinical trial. If a miRNAprofile is determined to be correlated with drug efficacy or drugtoxicity that may be relevant to whether that patient is an appropriatepatient for receiving the drug or for a particular dosage of the drug.

A diagnostic assay can be created based on the profiles that doctors canuse to identify individuals with a disease or who are at risk to developa disease. Alternatively, treatments can be designed based on miRNAprofiling.

C. Amplification

Many methods exist for evaluating miRNA levels by amplifying all or partof miRNA nucleic acid sequences such as mature miRNAs, precursor miRNAs,and primary miRNAs. Suitable nucleic acid polymerization andamplification techniques include reverse transcription (RT), polymerasechain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleicacid sequence-base amplification (NASBA), ligase chain reaction,multiplex ligatable probe amplification, invader technology (ThirdWave), rolling circle amplification, in vitro transcription (IVT),strand displacement amplification, transcription-mediated amplification(TMA), RNA (Eberwine) amplification, and other methods that are known topersons skilled in the art. In certain embodiments, more than oneamplification method may be used, such as reverse transcription followedby real time PCR (Chen et al., 2005 and/or U.S. patent application Ser.No. 11/567,082, filed Dec. 5, 2006, which are incorporated herein byreference in their entirety).

In PCR and q-PCR methods, for example, a set of primers is used for eachtarget sequence. In certain embodiments, the lengths of the primersdepends on many factors, including, but not limited to, the desiredhybridization temperature between the primers, the target nucleic acidsequence, and the complexity of the different target nucleic acidsequences to be amplified. In certain embodiments, a primer is about 15to about 35 nucleotides in length. In other embodiments, a primer isequal to or fewer than 15, 20, 25, 30, or 35 nucleotides in length. Inadditional embodiments, a primer is at least 35 nucleotides in length.

In a further aspect, a forward primer can comprise at least one sequencethat anneals to a target miRNA and alternatively can comprise anadditional 5′ noncomplementary region. In another aspect, a reverseprimer can be designed to anneal to the complement of a reversetranscribed miRNA. The reverse primer may be independent of the miRNAsequence, and multiple miRNAs may be amplified using the same reverseprimer. Alternatively, a reverse primer may be specific for a miRNA.

In some embodiments, two or more miRNAs or nucleic acids are amplifiedin a single reaction volume or multiple reaction volumes. In certainaspects, one or more miRNA or nucleic may be used as a normalizationcontrol or a reference nucleic acid for normalization. Normalization maybe performed in separate or the same reaction volumes as otheramplification reactions. One aspect includes multiplex q-PCR, such asqRT-PCR, which enables simultaneous amplification and quantification ofat least one miRNA of interest and at least one reference nucleic acidin one reaction volume by using more than one pair of primers and/ormore than one probe. The primer pairs comprise at least oneamplification primer that uniquely binds each nucleic acid, and theprobes are labeled such that they are distinguishable from one another,thus allowing simultaneous quantification of multiple miRNAs. MultiplexqRT-PCR has research and diagnostic uses, including but not limited todetection of miRNAs for diagnostic, prognostic, and therapeuticapplications.

A single combined reaction for q-PCR, may be used to: (1) decrease riskof experimenter error, (2) reduce assay-to-assay variability, (3)decrease risk of target or product contamination, and (4) increase assayspeed. The qRT-PCR reaction may further be combined with the reversetranscription reaction by including both a reverse transcriptase and aDNA-based thermostable DNA polymerase. When two polymerases are used, a“hot start” approach may be used to maximize assay performance (U.S.Pat. Nos. 5,411,876 and 5,985,619, each incorporated herein by referencein its entirety). For example, the components for a reversetranscriptase reaction and a PCR reaction may be sequestered using oneor more thermoactivation methods or chemical alteration to improvepolymerization efficiency (U.S. Pat. Nos. 5,550,044, 5,413,924, and6,403,341, each incorporated herein by reference in its entirety).

To assess the expression of microRNAs, real-time RT-PCR detection can beused to screen nucleic acids or RNA isolated from samples of interestand a related reference such as normal adjacent tissue (NAT) samples.

A panel of amplification targets is chosen for real-time RT-PCRquantification. The selection of the panel or targets can be based onthe results of microarray expression analyses, such as mirVana™ miRNABioarray V1, Ambion. In one aspect, the panel of targets includes one ormore miRNA described herein. One example of a normalization target is 5SrRNA and others can be included. Reverse transcription (RT) reactioncomponents are typically assembled on ice prior to the addition of RNAtemplate. Total RNA template is added and mixed. RT reactions areincubated in an appropriate PCR System at an appropriate temperature(15-70° C., including all values and ranges there between) for anappropriate time, 15 to 30 minutes or longer, then at a temperature of35 to 42 to 50° C. for 10 to 30 to 60 minutes, and then at 80 to 85 to95° C. for 5 minutes, then placed on wet ice. Reverse Transcriptionreaction components typically include nuclease-free water, reversetranscription buffer, dNTP mix, RT Primer, RNase Inhibitor, ReverseTranscriptase, and RNA.

PCR reaction components are typically assembled on ice prior to theaddition of the cDNA from the RT reactions. Following assembly of thePCR reaction components a portion of the RT reaction is transferred tothe PCR mix. PCR reaction are then typically incubated in an PCR systemat an elevated temperature (e.g., 95° C.) for 1 minute or so, then for anumber of cycles of denaturing, annealing, and extension (e.g., 40cycles of 95° C. for 5 seconds and 60° C. for 30 seconds). Results canbe analyzed, for example, with SDS V2.3 (Applied Biosystems). Real-timePCR components typically include Nuclease-free water, MgCl₂, PCR Buffer,dNTP mix, one or more primers, DNA Polymerase, cDNA from RT reaction andone or more detectable label.

Software tools such as NormFinder (Andersen et al., 2004) are used todetermine targets for normalization with the targets of interest andtissue sample set. For normalization of the real-time RT-PCR results,the cycle threshold (C_(t)) value (a log value) for the microRNA ofinterest is subtracted from the geometric mean C_(t) value ofnormalization targets. Fold change can be determined by subtracting thedC_(t) normal reference (N) from the corresponding dC_(t) sample beingevaluated (T), producing a ddC_(t)(T-N) value for each sample. Theaverage ddC_(t)(T-N) value across all samples is converted to foldchange by 2^(ddCt). The representative p-values are determined by atwo-tailed paired Student's t-test from the dC_(t) values of sample andnormal reference.

D. Nucleic Acid Arrays

Certain aspects of the present invention concern the preparation and useof miRNA arrays or miRNA probe arrays, which are ordered macroarrays ormicroarrays of nucleic acid molecules (probes) that are fully or nearlycomplementary or identical to a plurality of miRNA molecules orprecursor miRNA molecules and are positioned on a support or supportmaterial in a spatially separated organization. Macroarrays aretypically sheets of nitrocellulose or nylon upon which probes have beenspotted. Microarrays position the nucleic acid probes more densely suchthat up to 10,000 nucleic acid molecules can be fit into a regiontypically 1 to 4 square centimeters.

Representative methods and apparatus for preparing a microarray havebeen described, for example, in U.S. Pat. Nos. 5,143,854; 5,202,231;5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,432,049; 5,436,327; 5,445,934; 5,468,613;5,470,710; 5,472,672; 5,492,806; 5,503,980; 5,510,270; 5,525,464;5,527,681; 5,529,756; 5,532,128; 5,545,531; 5,547,839; 5,554,501;5,556,752; 5,561,071; 5,571,639; 5,580,726; 5,580,732; 5,593,839;5,599,695; 5,599,672; 5,610,287; 5,624,711; 5,631,134; 5,639,603;5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972; 5,695,940;5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; 5,837,196;5,871,928; 5,847,219; 5,876,932; 5,919,626; 6,004,755; 6,087,102;6,368,799; 6,383,749; 6,617,112; 6,638,717; 6,720,138, as well as WO93/17126; WO 95/11995; WO 95/21265; WO 95/21944; WO 95/35505; WO96/31622; WO 97/10365; WO 97/27317; WO 99/35505; WO 09923256; WO09936760; WO0138580; WO 0168255; WO 03020898; WO 03040410; WO 03053586;WO 03087297; WO 03091426; WO03100012; WO 04020085; WO 04027093; EP 373203; EP 785 280; EP 799 897 and UK 8 803 000; the disclosures of whichare all herein incorporated by reference. Moreover, a person of ordinaryskill in the art could readily analyze data generated using an array.Such protocols are disclosed above, and include information found in WO9743450; WO 03023058; WO 03022421; WO 03029485; WO 03067217; WO03066906; WO 03076928; WO 03093810; WO 03100448A1, all of which arespecifically incorporated by reference.

E. Hybridization

After an array or a set of miRNA probes is prepared and the miRNA in thesample is labeled, the population of target nucleic acids is contactedwith the array or probes under hybridization conditions, where suchconditions can be adjusted, as desired, to provide for an optimum levelof specificity in view of the particular assay being performed. Suitablehybridization conditions are well known to those of skill in the art andreviewed in Sambrook et al. (2001) and WO 95/21944. Of particularinterest in many embodiments is the use of stringent conditions duringhybridization. Stringent conditions are known to those of skill in theart.

VI. KITS

Any of the compositions or components described herein may be comprisedin a kit. In a non-limiting example, reagents for isolating miRNA,labeling miRNA, and/or evaluating a miRNA population using an array,nucleic acid amplification, and/or hybridization can be included in akit, as well reagents for preparation of samples from A subject. The kitmay further include reagents for creating or synthesizing miRNA probesor therapeutics. The kits will thus typically comprise, in suitablecontainer means, an enzyme for labeling the miRNA by incorporatinglabeled nucleotide or unlabeled nucleotides that are subsequentlylabeled. In certain aspects, the kit can include amplification reagents.In other aspects, the kit may include various supports, such as glass,nylon, polymeric beads, magnetic beads, and the like, and/or reagentsfor coupling any probes and/or target nucleic acids. It may also includeone or more buffers, such as pharmaceutical buffer, reaction buffer,labeling buffer, washing buffer, or a hybridization buffer, compoundsfor preparing the miRNA probes, and components for isolating miRNA.Other kits of the invention may include components for making a nucleicacid array comprising miRNA, and thus, may include, for example, a solidsupport.

Kits for implementing methods of the invention described herein arespecifically contemplated. In some embodiments, there are kits forpreparing miRNA for multi-labeling and kits for preparing miRNA probesand/or miRNA arrays. In these embodiments, kit comprise, in suitablecontainer means, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more of thefollowing: (1) poly(A) polymerase; (2) unmodified nucleotides (G, A, T,C, and/or U); (3) a modified nucleotide (labeled or unlabeled); (4)poly(A) polymerase buffer; and, (5) at least one microfilter; (6) labelthat can be attached to a nucleotide; (7) at least one miRNA probe; (8)reaction buffer; (9) a miRNA array or components for making such anarray; (10) acetic acid; (11) alcohol; (12) solutions for preparing,isolating, enriching, and purifying miRNAs or miRNA probes or arrays.Other reagents include those generally used for manipulating RNA, suchas formamide, loading dye, ribonuclease inhibitors, and DNase.

In specific embodiments, kits of the invention include an arraycontaining miRNA probes, as described in the application. An array mayhave probes corresponding to all known miRNAs of an organism or aparticular tissue or organ in particular conditions, or to a subset ofsuch probes. The subset of probes on arrays of the invention may be orinclude those identified as relevant to a particular diagnostic,therapeutic, or prognostic application. For example, the array maycontain one or more probes that is indicative or suggestive of (1) adisease or condition (neovascularization or aberrant angiogenesis), (2)susceptibility or resistance to a particular drug or treatment; (3)susceptibility to toxicity from a drug or substance; (4) the stage ofdevelopment or severity of a disease or condition (one aspect ofprognosis); (5) the likelihood of recurrence (one aspect of prognosis)and (6) genetic predisposition to a disease or condition.

For any kit embodiment, including an array, there can be nucleic acidmolecules that contain or can be used to amplify a sequence that is avariant of, identical to or complementary to all or part of any of SEQID NOs described herein. Any nucleic acid discussed above may beimplemented as part of a kit.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquotted. Where there is more than one component in the kit (labelingreagent and label may be packaged together), the kit also will generallycontain a second, third or other additional container into which theadditional components may be separately placed. However, variouscombinations of components may be comprised in a vial. The kits of thepresent invention also will typically include a means for containing thenucleic acids, and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow moldedplastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent or buffer. Itis envisioned that the solvent may also be provided in another containermeans. In some embodiments, labeling dyes are provided as a dried power.It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120,120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700,800, 900, 1000 μg or at least or at most those amounts of dried dye areprovided in kits of the invention. The dye may then be resuspended inany suitable solvent, such as DMSO.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which thenucleic acid formulations are placed, preferably, suitably allocated.The kits may also comprise a second container means for containing asterile, pharmaceutically acceptable buffer and/or other diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Such kits may also include components that facilitate isolation of thelabeled miRNA. It may also include components that preserve or maintainthe miRNA or that protect against its degradation. Such components maybe RNase-free or protect against RNases. Such kits generally willcomprise, in suitable means, distinct containers for each individualreagent or solution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented.

Kits of the invention may also include one or more of the following:Control RNA; nuclease-free water; RNase-free containers, such as 1.5 mltubes; RNase-free elution tubes; PEG dextran; ethanol; acetic acid;sodium acetate; ammonium acetate; guanidinium; detergent; nucleic acidsize marker; RNase-free tube tips; and RNase or DNase inhibitors.

It is contemplated that such reagents are embodiments of kits of theinvention. Such kits, however, are not limited to the particular itemsidentified above and may include any reagent used for the manipulationor characterization of miRNA.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 miRNAS Altered During Ischemic Retinopathy-InducedNeovascularization

To examine miRNA expression during retinal neovascularization, theinventors used a murine model of oxygen-induced, ischemic retinopathythat results in reproducible, proliferative retinal neovascularization(Smith et al., 1994.). C57BL/6J mice were placed in 75% oxygen onpostnatal day 7 and returned to room air on postnatal day 12. Onpostnatal day 15, mice were euthanized, eyes were removed, and retinasdissected. Small RNAs were isolated from retinas using the mirVana™miRNA Isolation Kit (cat. no. AM1561; Ambion Inc., Austin, Tex., USA)according to the manufacturer's instructions. Small RNAs (5 μg), pooledfrom ten ischemic retinas or from ten age-matched control retinas, wereidentified and quantified by microarray analysis. Microarrayhybridizations were performed by LC Sciences (Houston, Tex., USA). SmallRNAs from ischemic retinas were labeled with Cy3 and small RNAs fromcontrol retinas were labeled with Cy5 and hybridized to an array ofmurine microRNA (mmu-miR) probes with six redundant complementarysequences for each of the mmu-miR transcripts in the Sanger miRBaseRelease 7.0 (August 2005) (Griffiths-Jones et al., 2006). Red and greenfluorescence intensities were determined for each probe. Statisticalcomparisons were made between the mean differences in intensity for theprobes and the intensity differences for control probes. A p value of<0.01 was used to identify differentially expressed microRNAs (Table 1).Ten miRNAs (mmu-miR-451, -214, -424, -199a, -146, -106a, -350, -21,-218, and -148b) had increased expression during neovascularization ofischemic retinas, and six (mmu-miR-451, -214, -424, -199a, -146, and-106a) were increased more than two-fold. Nine miRNAs (mmu-miR-184, -31,-150, -409, -375, -129-5p, -124a, -29a, and -129-3p) had significantlyreduced expression during neovascularization of ischemic retinas, andthree (mmu-miR-184, -31, and -150) were decreased more than two-fold.

The miRNAs in Table 1 represent particularly useful therapeutic targetsfor treating conditions associated with vascularization

TABLE 1 MicroRNAs Differentially Expressed During Neovascularization ofMouse Retinas. Log Ratio miRNA (ischemia/control) miR-451 2.31 miR-2142.08 miR-424 2.01 miR-199a 1.52 miR-146 1.50 miR-106a 1.25 miR-350 0.95miR-21 0.91 miR-218 0.83 miR-148b 0.80 miR-184 −3.50 miR-31 −2.50miR-150 −2.01 miR-409 −0.81 miR-375 −0.80 miR-129-5p −0.75 miR-124a−0.65 miR-29a −0.63 miR-129-3p −0.61 Log ratio, the log(2) normalizedsignal for miRNA from ischemic mice divided by the log(2) normalizedsignal for miRNA from control mice. Positive log ratio values indicatemiRNAs with increased levels during neovascularization. Negative logratio values indicate miRNAs with decreased levels duringneovascularization.

Example 2 Quantification of miRNAS Altered During Neovascularization byORT-PCR

The inventors validated microarray data for specific miRNAs identifiedin Example 1. Using small RNAs isolated from retinas as described inExample 1 above, the inventors measured miRNA levels by qRT-PCR aspreviously described (Raymond et al., 2005), with minor modifications.Briefly, small RNAs were reverse-transcribed with SuperScript™ IIIReverse Transcriptase (Invitrogen Corp.; Carlsbad, Calif., USA) intocDNAs, using either (1) microRNA-specific primers containing a 36 bp 5′tail from C. elegans genomic sequence or (2) random hexamer-priming. FormiRNA amplifications, a miRNA-specific, 16-nucleotide upstream primerand a 19-nucleotide universal primer complementary to the C. eleganstail sequence were used. For standardization, a fragment of 5S rRNA wasamplified and quantified. Amplifications were performed in aLightCycler® (Roche Diagnostics Corp., Indianapolis, Ind., USA) usingthe LightCycler® FastStart DNA Master SYBR Green I reaction mix (Roche).For negative controls, cDNA generated from random hexamer priming wasused. The mean threshold cycle numbers (ΔCT) for mmu-miR-31, -150, and-184 amplified from ischemic and control retinas (n=5 for each) weredetermined, and the mean differences between the two values (ΔΔCT) werecalculated. ΔΔCT values ranged from about −1.0 (miR-31) to −2.5(miR-150) (FIG. 1A). Statistical comparisons confirmed that each of themiRs were significantly reduced in ischemic compared to control retinas(FIG. 1B).

Example 3 Predicted Gene Targets of MMU-MIR-184, MMU-MIR-31, andMMU-MIR-150

The inventors searched selected mRNA transcripts for sequences thatmight represent target binding sites for the microRNAs shown in Table 1above. Searches were performed in the 5′- and 3′-untranslated regions(UTR) from a group of genes encoding angiogenic factors and theirreceptors (VEGF, HGF, FGF-2, P1GF, IGF-1, TGF-beta, PDGF-A, PDGF-B,VG5q, Delta-like 4, SEMA3a, Sema3F, Notch4, EphA2, Roundabout,HIF-1alpha, Frizzed-4, SDF-1, PEDF, endostatin, vasohibin-1,thrombspodin-1, Ang-1, Ang-2, Flt-1, KDR, neuropilin-1, neuropilin-2,CXCR4, Tie1, Tie2, angiostatin). UTR sequences were obtained from theMus musculus genome sequence at the Ensembl Project (Birney et al.,2004) (at the World Wide Web address ensembl.org/index.html) and weresearched for possible microRNA target sites using several target siteprediction systems, including miRBase Targets (at World Wide Web addressmicrorna.sanger.ac.uk/targets/v4/), the miranda algorithm (John et al.,2005; Betel et al., 2008) (at the World Wide Web address microrna.org),RNAhybrid (Rehmsmeier et al., 2004) (at the internet addressbibiserv.techfak.uni-bielefeld.de/rnahybrid), and PicTar (Krek et al.,2005) (at internet address pictar.bio.nyu.edu). Previously reportedcriteria (Lewis et al., 2005; Sethupathy et al., 2006) for predictingmicroRNA targets were used as a guide and included (1) a conserved seedmatch, i.e., perfect Watson-Crick complementarity between nucleotides2-7 of the microRNA and a 6 nucleotide section of the 3′-UTR of the mRNAthat occurs at corresponding positions for multiple species, (2) aconserved anchoring A, which is an A nucleotide on the 3′-UTR justdownstream of the seed match that also occurs in the UTRs of the genesfrom multiple species, and (3) a conserved m8-t8 match, which is an A:Uor G:C match between the eighth nucleotide of the microRNA and thecorresponding position in the 3′-UTR in multiple species. Conservationanalyses between the mouse UTRs and corresponding UTRs for humans, rats,and dogs were performed using applications at the University of SantaCruz Genome Bioinformatics internet site, on the World Wide Web atgenome.ucsc.edu.

The inventors identified possible gene targets for mmu-miR-31, -150, and-184 (FIG. 2). Predicted target sequences for mmu-miR-31 were identifiedin Pdgfb, Hif1α, and Frizzled4. Predicted target sequences formmu-miR-150 were identified in Vegf, Pdgfb, Pdgfa, and Notch4. Predictedtarget sequences for mmu-miR-184 were identified in Frizzled4. Nopredicted target genes were identified for up-regulated miRNAs in Table1.

The predicted gene targets of mmu-miR-31, -150, and -184 representparticularly useful therapeutic targets for treating conditionsassociated with vascularization, through manipulation of theirexpression levels.

Example 4 Verification of miRNA Target Genes

The inventors utilized luciferase gene reporter assays to assess thevalidity of putative miRNA target regulatory sequences in Pdgfb,Frizzled4, Hif1α, Vegf, and Notch4. Putative miRNA target sequences wereamplified by PCR using retinal cDNA as template and amplificationprimers that included restriction sites for SpeI and HindIII. PCRproducts were cloned into a TOPO TA Cloning® vector (Invitrogen).Recombinant plasmid inserts (miRNA target sequences) were verified byDNA sequencing. For validation of miRNA target sequences in Pdgfb,Frizzled4, Hif1α, and Notch4, the cloned target sequences were excisedand subcloned into a luciferase microRNA expression reporter vector(pMIR-REPORT™, Ambion). For validation of miRNA target sequences inVegf, the inventors used a pGL2-CMV vector containing a 1.7 kb mouseVegf 3′-UTR coupled to a luciferase gene (R. C. Nichols, Department ofMicrobiology and Immunology, Dartmouth School of Medicine, Hanover,N.H.).

Luciferase reporter assays were conducted in human retinal pigmentepithelial cells (ARPE-19; American Type Culture Collection, Manassas,Va., USA) cultured in DMEM/F12 medium (HyClone; Logan, Utah, USA)supplemented with 10% fetal bovine serum. After reaching 70% confluence,cells were co-transfected with a synthetic microRNA (mmu-miR-31, -150,or -184; pre-miR™ miRNA Precursor Molecule; Ambion) (25 mmol), 1 μg ofreporter vector (pMIR-REPORT™ or pGL2-CMV) containing one of thepossible target 3′-UTRs, and 2.5 ng of Renilla luciferase vector(Promega Corp.; Madison, Wis., USA). For negative controls, ARPE-19cells were co-transfected with a scrambled synthetic microRNA (25 nmol),1 μg of reporter vector containing one of the possible target 3′-UTRs,and 2.5 ng of Renilla luciferase vector. After 48 hours, cells wereharvested and luciferase expression assays were performed using theDual-Luciferase® Reporter Assay System (Promega). Luciferase expressionassays were performed in triplicate for each miRNA/target 3′-UTRcombination. Statistical comparisons with negative control assays weremade by paired t-test.

Co-transfection of ARPE-19 cells with synthetic mmu-miR-31 and a pMIRvector with the Pdgfb or Hif1α 3′-UTR, significantly reduced luciferaselevels expressed by the reporter (FIG. 3A, 3C), verifying that Pdgfb andHif1α are targets for regulation by mmu-miR-31. Co-transfection withsynthetic mmu-miR-31 and a pMIR vector with the Frizzled4 3′-UTR had noeffect on luciferase levels from the reporter (FIG. 3B).

Co-transfection or ARPE-19 cells with synthetic mmu-miR-150 and a pMIRvector with the Pdgfb 3′-UTR or with synthetic mmu-miR-150 and apGL2-CMV vector with the Vegf 3′-UTR, significantly reduced luciferaselevels expressed by the reporter (FIG. 3D, 3E), verifying that Pdgfb andVegf are targets for regulation by mmu-miR-150. Co-transfection withsynthetic mmu-miR-150 and a pMIR vector with the Notch4 3′-UTR had noeffect on luciferase levels from the reporter (FIG. 3F).

Co-transfection or ARPE-19 cells with synthetic mmu-miR-184 and a pMIRvector with the Frizzled4 3′-UTR significantly reduced luciferase levelsexpressed by the reporter (FIG. 3B), verifying that Frizzled4 is atarget for regulation by mmu-miR-184.

Example 5 Effect of miRNAS on Target Gene Product Levels inNeovascularizing Retinas In Vivo

The inventors evaluated the effects of mmu-miR-31, -250, and -184 ontarget gene product levels in vivo, during retinal neovascularizationinduced by ischemic retinopathy. C57/BL6 mice were placed in 75% oxygenon postnatal day 7. On postnatal day 12, mice were returned to room airand given an intraocular injection of a synthetic experimental microRNA(0.005 μmol in 1 μl) (pre-miR™ miRNA Precursor Molecule; Ambion) in oneeye and a synthetic control microRNA (0.005 μmol in 1 μl; scrambledmicroRNA sequence) in the other eye. Synthetic miRNAs were injected witha Harvard Pump Microinjection System (Harvard Apparatus, Holliston,Mass., USA) and pulled glass micropipettes as previously described (Moriet al., 2001). Mice were euthanized at postnatal day 15, eyes wereremoved, and retinas were dissected and homogenized. Retinas were placedin 200 μl of lysis buffer (50 μl 1M Tris-HCl (pH 7.4), 50 μl of 10%sodium dodecyl sulfate, 5 μl of 100 nM phenylmethanesulfonyl, and 5 mlof sterilized, de-ionized water), homogenized, sonicated at 4° C. for 5seconds, and centrifuged at 10,000 g for 5 minutes at 4° C. The proteinconcentration of the supernatants was measured with a bicinchoninic acidprotein assay kit (Pierce; Rockford, Ill., USA). Retinal homogenateswere used to assess HIF-1α, PDGF-B, and Frizzled 4 by immunoblotanalysis and VEGF by both immunoblot analysis and ELISA. Forimmunoblots, 20 μg of protein were separated by SDS-PAGE on 8-12%acrylamide gels and transferred to a nitrocellulose membrane.Non-specific binding was blocked by incubation in Tris-buffered saline(TBS) containing 5% skim milk, and membranes were hybridized with 0.5μg/ml rabbit anti-mouse VEGF polyclonal antibody (Abcam Inc.; Cambridge,Mass., USA), rabbit anti-mouse Frizzled 4 antibody (R&D Systems Inc.;Minneapolis, Minn., USA), rabbit anti-mouse PDGF-B or HIF1-α antibody(Santa Cruz Biotechnology® Inc.; Santa Cruz, Calif., USA), or in TBScontaining 0.05% Tween 20 and 2.5% skim milk, at 4° C. overnight. Afterthree washes with TBS-Tween-20, the membrane was incubated inhorseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonalantibody (1:5000) (GE Healthcare Bio-Sciences Corp.; Piscataway, N.J.,USA). For signal development, membranes were incubated in ECL Plus™Western Blotting Detection Reagent (GE Healthcare Bio-Sciences) andexposed to X-ray film. ELISAs were performed using a Quantikine VEGFassay kit (R&D Systems Inc.) according to the manufacturer'sinstructions. Serial dilutions of recombinant VEGF were assayed togenerate a standard curve. The limit of detection was 125 pg/ml of VEGF.

Injection of synthetic mmu-miR-31 or -150, but not syntheticmmu-miR-184, caused a significant reduction in VEGF levels inhomogenates of neovascularizing retinas (FIG. 4A, FIG. 4B). Sincemmu-miR-31 does not target Vegf directly, it is likely that its effectoccurs indirectly through one of its actual targets, probably Hif1α.Injection of 1 μl containing 1.66 pmol each of the three syntheticmicroRNAs also caused a significant reduction in VEGF (FIG. 4A).Intraocular injection of synthetic mmu-miR-31 caused reductions inretinal HIF1-α and PDGF-B (FIG. 4B). Injection of synthetic mmu-miR-150caused reductions in PDGF-B and VEGF (FIG. 4B). Injections of syntheticmmu-miR-31 or synthetic mmu-miR-184 failed to cause reductions inFrizzled 4 protein in the retinas (FIG. 4B).

Example 6 miRNA Therapy in Mice with Oxygen-Induced Ischemic Retinopathy

Ischemic retinopathy was generated in mice as described above inExample 1. Mice were returned to room air at postnatal day 12 and givena 1 μl intraocular injection containing 2 μg of experimental syntheticmicroRNA in one eye or 2 μg of control synthetic microRNA in the othereye. Injections were given as described above in Example 5. On postnatalday 17, areas of neovascularization on the surfaces of the mouse retinaswere measured as previously described (Lima e Silva et al., 2007).Briefly, mice were given an intraocular injection of 1 μl of ratanti-mouse PECAM-1 antibody (BD Biosciences; San Jose, Calif., USA) andwere euthanized twelve hours after antibody injection. Mouse eyes weredissected and fixed in 10% formalin for 4 hours. Intact retinas weredissected, incubated for 40 minutes in a 1:500 dilution of goat anti-ratIgG conjugated with Alexa488 (Invitrogen), washed, and whole mounted.This technique provides selective staining of retinal neovascularizationon the surface of the retina (Shen et al., 2007). An observer, maskedwith respect to treatment groups, examined the slides by fluorescencemicroscopy and measured the area of neovascularization per retina usingcomputerized image analysis and Image-Pro® PLUS software (MediaCybernetics, Inc.; Bethesda, Md., USA).

Retinas from mouse eyes injected with negative control miRNA showedextensive neovascularization (FIG. 5A). In contrast, retinas from mouseeyes injected with 2 μg of synthetic mmu-miR-31, -150, or -184 showedvery little neovascularization on the surface of the retina (FIG. 5B).Computerized image analysis confirmed a significant reduction in meanarea of neovascularization per retina in eyes injected with syntheticmmu-miR-31, -150, or -184 (FIG. 6A, FIG. 6B, FIG. 6C) or with a mixtureof all three synthetic miRs (FIG. 6D) as compared to eyes injected withnegative control miRNA.

Example 7 miRNA Therapy in Mice with Choroidal Neovascularization

The inventors used a model of choroidal neovascularization to furtherevaluate miRNA therapy. The choroid, also known as the choroidea orchoroid coat, is the vascular layer of the eye lying between the retinaand the sclera (the fibrous, protective outer layer of the eye). Thechoroid provides oxygen and nourishment to the outer layers of theretina.

Choroidal neovascularization was generated by modification of apreviously described technique (To be, et al., 1998). Briefly, 4 to 5week old female C57BL/6J mice were anesthetized and the pupils weredilated with 1% tropicamide. Three burns of 532 nm diode laserphotocoagulation (75 μm spot size, 0.1 second duration, 120 mW) weredelivered to each retina using the slit lamp delivery system of anOcuLight GL Photocoagulator (IRIDEX Corp., Mountain View, Calif., USA).Burns were performed in the 9-, 12-, and 3-o'clock positions of theposterior pole of the retina. Immediately after laser treatment andagain on day 7, mice were given an intraocular injection of 1 μlcontaining 5 μmol of experimental synthetic microRNA or a mixture ofthree experimental synthetic microRNAs (1.67 μmol each mmu-miR-31, -150,and -184) in one eye and 5 μmol of a scrambled, synthetic microRNA(control) in the other eye. Fourteen days after laser treatment, micewere perfused with fluorescein-labeled dextran (2×10⁶ average MW)(Sigma-Aldrich, St. Louis, Mo., USA), and choroidal whole mounts wereprepared and examined by fluorescence microscopy. An observer, maskedwith respect to treatment group, used image analysis techniques tomeasure the area of choroidal neovascularization at each Bruch'smembrane rupture site (Shen et al., 2006; Lima e Silva et al., 2007).The three choroidal neovascularization areas within each eye wereaveraged to give one experimental value per eye. Statistical comparisonswere made by paired t-test.

Fluorescence microscopy observation revealed that eyes treated with amixture of three experimental miRNAs (mmu-miR-31, -150, and -184) (FIG.7A) had smaller choroidal neovascularization lesions than fellow eyestreated with the scrambled, control miRNA (FIG. 7B). Measurement of thearea of choroidal neovascularization by computerized image analysisrevealed that eyes treated with the mixture of experimental miRNAs had asignificant reduction in the mean area of choroidal neovascularizationat Bruch's membrane rupture sites when compared to eyes treated with thescrambled, control miRNA (FIG. 8A). Mouse eyes treated individually withmmu-miR-31 (FIG. 8B) or mmu-miR-150 (FIG. 8C) also exhibited reducedchoroidal neovascularization. Mouse eyes treated with mmu-miR-184displayed similar levels of choroidal neovascularization as thosetreated with the control miRNA (FIG. 8D).

Example 8 miRNAS Altered in RD1 Mouse Model of Neovascularization

The inventors examined miRNA expression changes in a second model ofneovascularization, consisting of a transgenic mouse strain (RD1) thatcontains a phosphodiesterase 6B mutation (Chang et al., 2002). RD1 miceexhibit early onset retinal degeneration wherein they undergo roddegeneration between postnatal day 10 (P10) and postnatal day 22 (P22)and cone degeneration between postnatal day 22 (P22) and postnatal day35 (P35).

To track changes in miRNA expression during the progression ofneo-vascularization in eyes of RD1 mice, animals were sacrificed at P5,P10, P15, P22, and P35, eyes were removed, and retinas dissected. SmallRNAs were isolated from retinas using the mirVana™ miRNA Isolation Kit(Ambion). To account for changes in miRNA expression during normal mousedevelopment, a set of control mice (strain C57) were sacrificed at P5,P10, P15, P22, and P35, eyes were removed, retinas dissected, and smallRNAs were isolated as for the RD1 mice.

miRNAs from all mice were purified using the flashPAGE™ FractionatorApparatus (cat. no. AM13100, Ambion) and labeled using the mirVana™miRNA Labeling Kit (cat. no. AM1562, Ambion). Labeled miRNAs werehybridized to mirVana™ miRNA Bioarrays (Ambion). Signals for eachmiRNA-specific probe were background-subtracted, normalized, andconverted to log(2) scale. The normalized expression data for each miRNAat each time point were compared to the corresponding data for the sameanimals at P10 (the day of retinal degeneration onset).

The inventors observed differential expression of specific miRNAs duringretinal neovascularization in RD1 mice as compared to their expressionin the control mice (Table 2). Forty-seven miRNAs were observed to besignificantly differentially expressed, at one or both of P22 and P35,in the RD1 mice relative to the C57 control mice—mmu-miR-10b, -96, -183,-184, -16, -182, -191, -29c, -181c, -129-3p, -335, -210, -512-3p, -132,-500, -339, -511, -26b, -30b, and -15a and ambi-miR-7026 were allsignificantly decreased in mouse retinas undergoing neovascularization(Table 2) and mmu-miR-205, -106a, -365, -299-5p, -200a, -351, -329,-122a, -20a, -350, -520h, -142-5p, -203, -211, -145, -93, -192, -106a,-201, -18a, -17-5p, -106b, and -223, mmu-let-7b, ambi-miR-7079, andambi-miR-7085, were all significantly increased in mouse retinasundergoing neo-vascularization (Table 2). The miRNAs in Table 2represent particularly useful therapeutic targets for treatingconditions associated with vascularization.

TABLE 2 MicroRNAs Differentially Expressed During Neovascularization ofRetinas in RD1 Mice. Log Ratio Log Ratio RD1-C57 RD1-C57 miRNA (P22)(P35) miR-10b −2.525 −2.792 miR-96 −2.404 −2.58 miR-183 −1.726 −1.938miR-184 −2.345 −1.829 miR-16 −1.769 −1.24 miR-182 −1.983 −1.171 miR-191−1.006 −1.153 miR-29c −2.195 −1.03 miR-181c −1.096 −1.017 miR-129-3p−0.428 −1.015 miR-335 −1.005 −0.967 ambi-miR-7026 0.461 −0.88 miR-210−0.672 −0.862 miR-512-3p −0.132 −0.845 miR-132 −0.229 −0.832 miR-500−0.395 −0.77 miR-339 −0.295 −0.712 miR-511 −0.124 −0.695 miR-26b −1.375−0.689 miR-30b −1.35 −0.685 miR-15a −1.01 −0.601 miR-205 2.588 2.601miR-106a 0.957 2.585 miR-365 2.332 2.359 miR-299-5p 2.408 2.318ambi-miR-7079 2.495 2.302 miR-200a 2.104 2.238 miR-351 1.956 2.083miR-329 0.111 1.367 miR-122a 0.958 1.35 miR-20a 0.755 1.266 miR-350 0.591.258 miR-520h 1.094 1.247 miR-142-5p 0.153 1.157 miR-203 0.637 1.142miR-211 0.593 1.093 miR-145 0.213 1.072 let-7b 0.364 1.06 miR-93 0.9691.045 miR-192 0.319 1.001 miR-106a 0.83 0.929 miR-201 0.704 0.916miR-18a 0.611 0.907 miR-17-5p 0.741 0.864 ambi-miR-7085 0.667 0.848miR-106b 0.549 0.842 miR-223 0.527 0.819 Positive log ratio valuesindicate miRNAs with increased levels during neovascularization.Negative log ratio values indicate miRNAs with decreased values duringneovascularization. P22; post-natal day 22. P35; post-natal day 35.

Example 9 Predicted MicroRNA Targets

The miRNAs that were observed to be significantly differentiallyexpressed in the retinas of mice undergoing neovascularization wereevaluated for their capacity to potentially regulate genes associatedwith angiogenesis. Gene target predictions were performed by searchingwithin the 3′UTRs of 23 angiogenesis-related genes (LRP6, VEGFA, VEFC,VEGF-R, EFEMP1, ECGF1, ELOVL4, EREG, FGF1, FGF2, IGF1, JAG1, NRP1, NRP2,PGF, RS1, RDS, MMP2, MMP9, TIMP1, TIMP2, TIMP3, TLR4) for sites that areperfectly complementary with the core sequences of the selected miRNAs.Predicted targets for miRNAs are shown in Table 3. Many of the miRNAswere predicted to have multiple potential target sites within a single3′UTR. The number of predicted target interactions is denoted inparentheses. The average number of angiogenesis-related target sites forthe neovascularization-associated miRNAs was 4.64, with a range of 0-12.Based on this analysis, it is apparent that the altered expression ofone or several of these miRNAs can profoundly influence the expressionlevels of genes that are known to inhibit or activate blood vesselgrowth.

TABLE 3 Predicted target genes of miRNAs altered duringneovascularization. Genes with multiple predicted target interactionsites have the number of such sites indicated in parentheses followingthe gene name. Some genes had no targets predicted by the methodsdescribed in Example 9. miRNA Predicted Target Genes miR-10b NRP2 RS1miR-96 miR-183 LRP6 MMP9 TIMP3 NRP1 miR-16/miR-15a VEGFA LRP6 NRP2 (3)FGF2 (4) RDS (2) RS1 miR-182 VEGFR miR-191 TIMP2 ELOVL4 RDS RS1miR-29c/miR-29a VEGFA MMP2 EREG (2) IGF1 (2) ELOVL4 EFEMP1 RDS miR-181cTIMP3 (2) IGF1 RDS miR-129-3p NRP1 NRP2 miR-335 FGF2 RDS (2) miR-210NRP2 miR-512-3p TIMP2 FGF1 FGF2 miR-132 RS1 miR-500 VEGFR (2) TIMP2 NRP1NRP2 FGF2 (2) ANPEP miR-339 VEGFA (2) MMP2 NRP2 CFB RS1 miR-511 VEGFAVEGFC MMP2 NRP1 (2) FGF2 TLR4 (3) VMD2 ELOVL4 (2) miR-26b JAG1 EREG IGF1TLR4 EFEMP1 miR-30b TIMP3 NRP1 miR-31 TIMP2 IGF1 TLR4 (2) miR-150 VEGFANRP1 FGF1 FGF2 (2) EREG IGF1 RS1 miR-129-5p APOE TIMP2 NRP1 JAG1 FGF2ANGPT1 IGF1 (4) RDS miR-124a MMP2 PGF NRP1 NRP2 JAG1 miR-451 EREG FBLN5miR-214 VEGFR PGF (3) TIMP3 NRP1 JAG1 CFB FBLN5 RDS miR-424 VEGFA LRP6NRP2 (3) FGF2 (4) RDS (2) RS1 miR-199a JAG1 miR-146 TIMP3 NRP2 (2) JAG1FGF2 CFH TLR4 miR-106a/20a/17- VEGFA MMP2 (2) TIMP2 FGF2 (2) EREG 5pmiR-21 TIMP3 (2) JAG1 FGF1 FGF2 CFH TLR4 miR-218 TIMP2 JAG1 IGF1 RDSmiR-148b NRP1 FGF2 ANGPT2 IFG1 VMD2 miR-205 VEGFA FGF1 FGF2 miR-365 EREGTLR4 EFEMP1 miR-299-5p VEGFR FGF1 miR-200a VEGFR NRP1 NRP2 (2) FBLN5miR-329 TIMP3 NRP2 TLR4 miR-122a TLR4 RS1 miR-520h VEGFA MMP2 PGF FGF2(2) EREG ANGPT1 TLR4 miR-142-5p NRP1 JAG1 FGF2 ANGPT2 IGF1 (2) miR-203TIMP3 NRP1 NRP2 FGF2 EREG TLR4 (2) ABCA4 miR-211 MMP9 NRP2 FGF1 (2)ANGPT1 ELOVL4 RDS miR-145 NRP1 NRP2 ANGPT2 IGF1 TLR4 ELOVL4 let-7b IGF1ELOVL4 miR-93 VEGFA MMP2 (2) TIMP2 FGF2 (2) EREG miR-192 EREG IGF1miR-18a IGF1 miR-223 TIMP2 FGF2 (2) ABCA4 EFEMP1 miR-409 miR-375 miR-184

To determine if the number of angiogenesis target sites for the variousmiRNAs was significant, the inventors randomly selected twelve miRNAsand used the same list of genes and methodology to select potentialangiogenesis related target genes. The average number of target sitesfor the randomly selected miRNAs was 2.7 or almost two fewer than wasobserved for the neovascularization-related miRNAs. The Student's t-testwas used to calculate a p-value of 0.01, suggesting that the variance inthe number of target gene predictions for the neovascularizationassociated miRNAs and the randomly-selected miRNAs is significant. Afurther indication of the significant difference between the lists ofmiRNAs is that only one of the randomly selected miRNAs was predicted tohave at least two target sites for any single angiogenesis related gene.

TABLE 4 Predicted target genes of randomly selected miRNAs. Genes withmultiple predicted target interaction sites have the number of suchsites indicated in parentheses following the gene name. Some genes hadno targets predicted by the methods described in Example 9. miRNAPredicted Target Genes miR-24 VEGFA TIMP2 NRP1 NRP2 EREG RDS miR-26aJAG1 EREG IGF1 TLR4 EFEMP1 miR-103 VEGFA NRP2 FGF2 FGF2 TLR4 RS1 miR-143NRP2 FGF1 miR-9 EREG ANGPT2 ELOVL4 miR-124 MMP2 PFG NRP1 NRP2 JAG1miR-219-5p TIMP3 TLR4 miR-377 VEGFA TIMP3 NRP2 (2) JAG1 IGF1 ABCA4miR-384 TIMP2 IGF1 miR-7 FBLN5 TLR4 miR-28-5p PGF ECGF1 RS1 miR-601 NRP2miR-890 TIMP3 (2) miR-99a miR-202 miR-323-50 miR-126

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for reducing vascularization in a subject or tissuecomprising administering to the subject or tissue in need of such areduction, in an amount sufficient to reduce vascularization, one ormore nucleic acid molecules comprising a nucleic acid sequence 17 to 110nucleotides in length that is at least 80% identical to miR-31.
 2. Themethod of claim 1, wherein the nucleic acid is administered topically,enterally, parenterally or intravitreally.
 3. The method of claim 1,wherein the nucleic acid molecule is an RNA.
 4. The method of claim 3,wherein the RNA comprises a complementary RNA region that hasnoncomplementarity with the miRNA in the last 1, 2, 3, 4, and/or 5nucleotides of the complementary region.
 5. The method of claim 4,wherein the RNA comprises a hairpin structure.
 6. The method of claim 1,wherein the RNA is 17 to 35 nucleotides in length and comprises two RNAstrands, wherein the complementary strand is 1) between 60 and 90%complementary to the miRNA sequence or 2) has a nucleotide analog ormodified nucleotide.
 7. The method of claim 6, wherein the RNA moleculeincludes a nucleotide analog or a modified nucleotide in thecomplementary strand.
 8. The method of claim 1, wherein the nucleic acidmolecule is a DNA molecule or is produced from a DNA molecule.
 9. Amethod for reducing vascularization in a subject or tissue comprisingadministering to the subject or tissue in need of such a reduction, inan amount sufficient to reduce vascularization, a vector comprising anucleic acid molecule encoding a nucleic acid sequence that comprises asequence that is at least 80% identical to miR-31.
 10. The method ofclaim 1, wherein the subject has, is at risk of developing, or issuspected of having ocular or retinal/choroidal neovascular diseases,cancer, diabetic nephropathy, rheumatoid arthritis, atheroscleroticplaques, endometriosis, Crohn's disease, uterine fibroids, benignprostatic hyperplasia, or psoriasis.
 11. The method of claim 6, whereinthe modified nucleotide is a sugar modification.
 12. The method of claim6, wherein the complementary strand comprises a replacement group forthe phosphate or hydroxyl group.
 13. The method of claim 9, wherein thevector is comprised in a lipid or viral delivery vehicle.
 14. The methodof claim 9, wherein the vector is a viral vector.
 15. The method ofclaim 9, wherein the vector is administered topically, enterally,parenterally or intravitreally.
 16. The method of claim 9, wherein thesubject has, is at risk of developing, or is suspected of having ocularor retinal/choroidal neovascular diseases, cancer, diabetic nephropathy,rheumatoid arthritis, atherosclerotic plaques, endometriosis, Crohn'sdisease, uterine fibroids, benign prostatic hyperplasia, or psoriasis.